Oxidative retinal diseases

ABSTRACT

Some aspects of the invention provide for a method of treating Wet and/or Dry Age-related Macular Degeneration, Retinitis Pigmentosa, Diabetic Retinopathy, cataracts, and/or Stargardt Disease using polyunsaturated fatty acids which are modified in certain positions to attenuate oxidative damage by Reactive Oxygen Species (ROS) and/or suppress the rate of formation of reactive products and toxic compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 61/479,281, filed Apr. 26, 2011; which is herebyexpressly incorporated by reference in its entirety.

BACKGROUND

1. Field

Isotopically modified polyunsaturated fatty acids (“PUFAs”) and othermodified PUFAs for treating certain diseases, particularly Wet and DryAge-related Macular Degeneration (AMD); Retinitis Pigmentosa (RP);Diabetic Retinopathy (DR); Cataracts; and Stargardt Disease (SD).

2. Description of the Related Art

Oxidative stress is implicated in a wide variety of diseases such asmitochondrial diseases, neurodegenerative diseases, inborn error's ofmetabolism, diabetes, diseases of the eye, kidney diseases, liverdiseases, and cardiac diseases. Specifically, such diseases include butare not limited to Wet and Dry Age-related Macular Degeneration (AMD);Retinitis Pigmentosa (RP); Diabetic Retinopathy (DR); Cataracts; andStargardt Disease (SD). See, for example, U.S. Patent Application Nos.:20110046219 and 20080275005 and the references cited therein.

While the number of diseases associated with oxidative stress arenumerous and diverse, it is well established that oxidative stress iscaused by disturbances to the normal redox state within cells. Animbalance between routine production and detoxification of reactiveoxygen species (“ROS”) such as peroxides and free radicals can result inoxidative damage to cellular structures and machinery. Under normalconditions, potentially important sources of ROSs in aerobic organismsis the leakage of activated oxygen from mitochondria during normaloxidative respiration. Additionally, it is known that macrophages andenzymatic reactions also contribute to the generation of ROSs withincells. Because cells and their internal organelles are lipidmembrane-bound, ROSs can readily contact membrane constituents and causelipid oxidation. Ultimately, such oxidative damage can be relayed toother biomolecules within the cell, such as DNA and proteins, throughdirect and indirect contact with activated oxygen, oxidized membraneconstituents, or other oxidized cellular components. Thus, one canreadily envision how oxidative damage may propagate throughout a cellgive the mobility of internal constituents and the interconnectedness ofcellular pathways.

Lipid-forming fatty acids are well-known as one of the major componentsof living cells. As such, they participate in numerous metabolicpathways, and play an important role in a variety of pathologies.Polyunsaturated Fatty Acids (“PUFAs”) are an important sub-class offatty acids. An essential nutrient is a food component that directly, orvia conversion, serves an essential biological function and which is notproduced endogenously or in large enough amounts to cover therequirements. For homeothermic animals, the two rigorously essentialPUFAs are linoleic (cis,cis-9,12-Octadecadienoic acid;(9Z,12Z)-9,12-Octadecadienoic acid; “LA”; 18:2; n-6) and alpha-linolenic(cis,cis,cis-9,12,15-Octadecatrienoic acid;(9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid; “ALA”; 18:3; n-3) acids,formerly known as vitamin F (Cunnane S C. Progress in Lipid Research2003; 42:544-568). LA, by further enzymatic desaturation and elongation,is converted into higher n-6 PUFAs such as arachidonic (AA; 20:4; n-6)acid; whereas ALA gives rise to a higher n-3 series, including, but notlimited to, eicosapentaenoic acid (EPA; 20:5; n-3) and docosahexaenoic(DHA; 22:6; n-3) acid (Goyens P L. et al. Am. J. Clin. Nutr. 2006;84:44-53). Because of the essential nature of certain PUFAs or PUFAprecursors, there are many known instances of their deficiency and theseare often linked to medical conditions. Furthermore, many PUFAsupplements are available over-the-counter, with proven efficiencyagainst certain ailments (See, for example, U.S. Pat. No. 7,271,315 andU.S. Pat. No. 7,381,558).

PUFAs endow mitochondrial membranes with appropriate fluidity necessaryfor optimal oxidative phosphorylation performance. PUFAs also play animportant role in initiation and propagation of the oxidative stress.PUFAs react with ROS through a chain reaction that amplifies an originalevent (Sun M, Salomon R G, J. Am. Chem. Soc. 2004; 126:5699-5708).However, non-enzymatic formation of high levels of lipid hydroperoxidesis known to result in several detrimental changes. Indeed, Coenzyme Q10has been linked to increased PUFA toxicity via PUFA peroxidation and thetoxicity of the resulting products (Do T Q et al, PNAS USA 1996;93:7534-7539). Such oxidized products negatively affect the fluidity andpermeability of their membranes; they lead to oxidation of membraneproteins; and they can be converted into a large number of highlyreactive carbonyl compounds. The latter include reactive species such asacrolein, malonic dialdehyde, glyoxal, methylglyoxal, etc.(Negre-Salvayre A, et al. Brit. J. Pharmacol. 2008; 153:6-20). But themost prominent products of PUFA oxidation are alpha, beta-unsaturatedaldehydes such as 4-hydroxynon-2-enal (4-HNE; formed from n-6 PUFAs likeLA or AA), 4-hydroxyhex-2-enal (4-HHE; formed from n-3 PUFAs like ALA orDHA), and corresponding ketoaldehydes (Esterfbauer H, et al. Free Rad.Biol. Med. 1991; 11:81-128; Long E K, Picklo M J. Free Rad. Biol. Med.2010; 49:1-8). These reactive carbonyls cross-link (bio)moleculesthrough Michael addition or Schiff base formation pathways, and havebeen implicated in a large number of pathological processes (such asthose introduced above), age-related and oxidative stress-relatedconditions, and aging. Importantly, in some cases, PUFAs appear tooxidize at specific sites because methylene groups of 1,4-diene systems(the bis-allylic position) are substantially less stable to ROS, and toenzymes such as cyclogenases and lipoxygenases, than allylic methylenes.

We have now discovered that oxidation resistant PUFAs, PUFA mimetics,PUFA pro-drugs and/or fats containing oxidation resistant PUFAs and PUFAmimetics are useful for mitigating and/or treating oxidative retinaldiseases.

SUMMARY

Some embodiments provide a method of treating or inhibiting theprogression of an oxidative retinal diseases, comprising administeringan effective amount of a polyunsaturated substance to a Wet or DryAge-related Macular Degeneration (AMD), Retinitis Pigmentosa (RP),Diabetic Retinopathy (DR), Cataracts, or Stargardt Disease (SD) patientin need of treatment, wherein the polyunsaturated substance ischemically modified such that one or more bonds are stabilized againstoxidation, wherein the polyunsaturated substance or a polyunsaturatedmetabolite thereof comprising said one or more stabilized bonds isincorporated into the patient's body following administration.

In some embodiments, the polyunsaturated substance is a nutritionelement. In other embodiments, the nutrition element is a fatty acid, afatty acid mimetic, and/or a fatty acid pro-drug. In other embodiments,the nutrition element is a triglyceride, a diglyceride, and/or amonoglyceride comprising a fatty acid, a fatty acid mimetic, and/or afatty acid pro-drug. In some embodiments, the fatty acid, fatty acidmimetic, or fatty acid pro-drug is stabilized at one or more bis-allylicpositions. In other embodiments, the stabilization comprises at leastone ¹³C atom or at least one deuterium atom at a bis-allylic position.In some embodiments, the stabilization comprises at least two deuteriumatoms at one or more bis-allylic position. In other embodiments, thestabilization utilizes an amount of isotopes that is above thenaturally-occurring abundance level. In some embodiments, thestabilization utilizes an amount of isotopes that is significantly abovethe naturally-occurring abundance level of the isotope.

In some embodiments, the fatty acid, fatty acid mimetic, or fatty acidpro-drug has an isotopic purity of from about 20%-99%. In otherembodiments, the isotopically stabilized fatty acids, fatty acidmimetics, or fatty acid pro-drugs are administered to a patient alongwith non-stabilized fatty acids, fatty acid mimetics, or fatty acidpro-drugs. In some embodiments, the isotopically stabilized fatty acids,fatty acid mimetics, or fatty acid pro-drugs comprise between about 1%and 100%, between about 5% and 75%, between about 10% and 30%, or about20% or more of the total amount of fatty acids, fatty acid mimetics, orfatty acid pro-drugs administered to the patient. In some embodiments,the patient ingests the fatty acid, fatty acid mimetic, or fatty acidpro-drug. In some embodiments, a cell or tissue of the patient maintainsa sufficient concentration of the fatty acid, fatty acid mimetic, fattyacid pro-drug, triglyceride, diglyceride, and/or monoglyceride toprevent autooxidation of the naturally occurring polyunsaturated fattyacid, mimetic, or ester pro-drug. In some embodiments, the stabilizationutilizes an amount of isotope that is significantly above thenaturally-occurring abundance level of said isotope.

In some embodiments, the method utilizes a fatty acid, fatty acidmimetic, or fatty acid pro-drug that is an omega-3 fatty acid and/or anomega-6 fatty acid. In other embodiments, the fatty acid selected fromthe group consisting of 11,11-D2-linolenic acid, 14,14-D2-linolenicacid, 11,11,14,14-D4-linolenic acid, 11,11-D2-linoleic acid,14,14-D2-linoleic acid, 11,11,14,14-D4-linoleic acid, 11-D-linolenicacid, 14-D-linolenic acid, 11,14-D2-linolenic acid, 11-D-linoleic acid,14-D-linoleic acid, and 11,14-D2-linoleic acid. In other embodiments,the fatty acids are further stabilized at a pro-bis-allylic position. Insome embodiments, the fatty acid is alpha linolenic acid, linoleic acid,gamma linolenic acid, dihomo gamma linolenic acid, arachidonic acid,and/or docosatetraenoic acid. In some embodiments, the fatty acid isincorporated into the mitochondrial membrane. In other embodiments, thefatty acid pro-drug is an ester. In some embodiments, the ester is atriglyceride, diglyceride, or monoglyceride.

Some embodiments further comprise co-administering an antioxidant. Insome embodiments, the antioxidant is Coenzyme Q, idebenone, mitoquinone,or mitoquinol. In other embodiments, the antioxidant is amitochondrially-targeted antioxidant. In some embodiments, theantioxidant is a vitamin, vitamin mimetic, or vitamin pro-drug. In otherembodiments, the antioxidant is a vitamin E, vitamin E mimetic, vitaminE pro-drug, vitamin C, vitamin C mimetic, and/or vitamin C pro-drug.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. (1A) ROS-driven oxidation of PUFAs; (1B) formation oftoxic carbonyl compounds.

FIGS. 2A and 2B. ¹H- and ¹³C-NMR analysis of deuterated PUFAs describedin Examples 1-4.

FIG. 3. Sensitivity of coq null mutants to treatment with linolenic acidis abrogated by isotope-reinforcement. Yeast coq3, coq7 and coq9 nullmutants were prepared in the W303 yeast genetic background (WT). Yeaststrains were grown in YPD medium (1% Bacto-yeast extract, 2%Bacto-peptone, 2% dextrose) and harvested while in log phase growth(OD_(600 nm)=0.1-1.0). Cells were washed twice with sterile water andresuspended in phosphate buffer (0.10 M sodium phosphate, pH 6.2, 0.2%dextrose) to an OD_(600 nm)=0.2. Samples were removed and 1:5 serialdilutions starting at 0.20 OD/ml were plated on YPD plate medium, toprovide a zero time untreated control (shown in top left panel). Thedesignated fatty acids were added to 200 μM final concentration to 20 mlof yeast in phosphate buffer. At 2 h, 4 h, and 16 h samples wereremoved, 1:5 serial dilutions prepared, and spotted onto YPD platemedium. Pictures were taken after 2 days of growth at 30° C. This panelis representative of two independent assays, performed on differentdays.

FIG. 4. Yeast coq mutants treated with isotope-reinforced D4-linolenicacid are resistant to PUFA-mediated cell killing. The fatty acidsensitive assay was performed as described in FIG. 6-1, except that 100μl aliquots were removed at 1, 2, and 4 h and, following dilution,spread onto YPD plates. Pictures were taken after 2 to 2.5 days, and thenumber of colonies counted. Yeast strains include Wild type (circles),atp2 (triangles), or coq3 (squares); Fatty acid treatments include oleicC18:1 (solid line), linolenic, C18:3, n-3 (dashed line) or11,11,14,14-D4-linolenic, C18:3, n-3, (dotted line).

FIG. 5. Separation and detection of fatty acid methyl ester (FAME)standards by GC-MS. FAMEs were prepared as described (Moss C W, LambertM A, Merwin W H. Appl. Microbiol. 1974; 1, 80-85), and the indicatedamounts of free fatty acids and 200 μg of C17:0 (an internal standard)were subjected to methylation and extraction. Samples analyses wereperformed on an Agilent 6890-6975 GC-MS with a DB-wax column (0.25 mm×30m×0.25-m film thickness) (Agilent, catalog 122-7031).

FIG. 6. Uptake of exogenously supplied fatty acids by yeast. WT (W303)yeast were harvested at log phase and incubated in the presence of 200μM of the designated fatty acid for either 0 or 4 h. Yeast cells wereharvested, washed twice with sterile water and then subjected toalkaline methanolysis and saponification, and lipid extraction asdescribed (Moss C W, Lambert M A, Merwin W H. Appl. Microbiol. 1974; 1,80-85; (Shaw, 1953 Shaw, W. H. C.; Jefferies, J. P. Determination ofergosterol in yeast. Anal Chem 25:1130; 1953). Each designated fattyacid is given as μg per OD_(600 nm) yeast, and was corrected for therecovery of the C17:0 internal standard.

FIG. 7. Kinetics of O₂ consumption accompanied the oxidation of 0.71 MLA (plots 1 and 2) and 0.71 M D2-LA (plot 3) in chlorobenzene initiatedby 40 mM AMVN at 37° C. Plot 2—0.23 mM HPMC was added to 0.71 M LA.

FIG. 8. Dependence of the rate of oxidation of the mixture of LA andD2-LA in chlorobenzene solution on mixture composition. Conditions:[LA]+[11,11-d₂-LA]=0.775 M; [AMVN]=0.0217 M; 37° C.R_(IN)=(1.10±0.08)×10⁻⁷ M/sec.

FIG. 9. Isotope reinforcement at the bis-allylic position ofpolyunsaturated fatty acids attenuates lipid autoxidation. Wild-type,yeast Q-less coq3, or respiratory deficient cor1 null mutants wereincubated in the presence of 200 μM of LA and D2-LA at different ratiosof PUFAs. Serial dilutions (1:5) starting at 0.2 OD/ml were spotted onYPD solid plate medium. A zero-time untreated control is shown on thetop left. Growth at 30° C.

FIG. 10. Isotope reinforcement at the bis-allylic position ofpolyunsaturated fatty acids attenuates lipid autoxidation. Wild-type,yeast Q-less coq3, or respiratory deficient cor1 null mutants wereincubated in the presence of 200 μM of ALA and D4-LA at different ratiosof PUFAs. Serial dilutions (1:5) starting at 0.2 OD/ml were spotted onYPD solid plate medium. Growth at 30° C.

FIG. 11. Chromatograms of the yeast extracts subjected to GC-MSanalyses. The different traces represent the 0 and 4 h incubations,respectively. The peak area of Each FAME (C18:1, C18:3 and D4-linolenic)was divided by the peak area of the C17:0 standard, quantified with acalibration curve. The endogenous 16:0 and 16:1 change very little,while the exogenously added fatty acids increased significantly.

FIG. 12. Survival of H- and D-PUFA treated MVEC cells after acuteintoxication by paraquat. For all cell types tested, D-PUFA hadprotective effect compared to controls, similar to that shown on Figurefor MVEC cells.

FIG. 13. Animal dosage studies of 1:1 D2-LA/D4-ALA indicating tissueenrichment with deuterium.

FIG. 14. Animal dosage studies of 1:1 D2-LA/D4-ALA comparing any changesin fat distribution.

FIG. 15. Animal dosage studies of 1:1 D2-LA/ALA indicating tissueenrichment with deuterium.

FIG. 16. Control liver fat profile after 90-day animal dosage study.

FIG. 17. Animal dosage studies of 1:1 D2-LA/D4-ALA indicating liver fatprofile and enrichment with deuterium.

FIG. 18. Liver fat profile after 90-day animal dosage study with D2-LA.

FIG. 19. Animal dosage studies of 1:1 D2-LA/D4-ALA indicating brain fatprofile and enrichment with deuterium.

FIG. 20. Animal dosage studies of 1:1 D2-LA/ALA indicating brain fatprofile and enrichment with deuterium.

FIG. 21. Control brain fat profile after 90-day animal dosage study.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As used herein, abbreviations are defined as follows:

-   αLnn Alpha-linolenic acid-   4-HHE or HHE 4-Hydroxyhex-2-enal-   4-HNE 4-Hydroxynon-2-enal (4-HNE; formed from n-6 PUFAs like LA or    AA)-   AA Arachidonic (AA; 20:4; n-6) acid-   AcOH Acetic acid-   ALA Alpha-linolenic acid-   AMD Age-related macular degeneration-   AMVN 2,2′-Azobis(2,4-dimethylvaleronitrile)-   D- Deuterated-   D1 Mono-deuterated-   D2 Di-deuterated-   D2-LA Di-deuterated linoleic acid-   D3 Tri-deuterated-   D4 Tetra-deuterated-   D5 Penta-deuterated-   D6 Hexa-deuterated-   DHA Docosahexaenoic (22:6; n-3) acid-   DMF Dimethylformamide-   DR Diabetic Retinopathy-   EPA Eicosapentaenoic (20:5; n-3) acid-   EtOAc Ethyl acetate-   EtOH Ethanol-   FAME Fatty acid methyl ester-   HPMC 6-Hydroxy-2,2,5,7,8-pentamethylbenzochroman-   H-PUFA Non-deuterated polyunsaturated fatty acid-   IP Intraperitoneal-   IR Infrared-   KIE Kinetic isotope effect-   LA Linoleic acid-   LDL Low-density lipoprotein-   MDA Malondialdehyde-   MPTP 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-   MVEC Microvascular endothelium-   NINCDS Neurological and Communicative Disorders and Stroke-   PUFA(s) Polyunsaturated fatty acid(s)-   R_(IN) Rate of initiation-   ROS Reactive oxygen species-   R_(OX) Rate of oxidation-   RP Retinitis pigmentosa-   RPE Retinal pigment epithelium-   SD Stargardt Disease-   SNOMED Systematized Nomenclature of Medicine-   SOD Superoxide dismutase-   TDMS Toxicology Data Management System-   TH Tyrosine hydroxylase-   THF Tetrahydrofuran-   TLC Thin layer chromatography-   V-SMOW Vienna standard mean ocean water-   WT Wild type-   YPD Medium containing 1% Bacto-yeast extract, 2% Bacto-peptone, 2%    dextrose

Wet and Dry Age-related Macular Degeneration (AMD); Retinitis Pigmentosa(RP); Diabetic Retinopathy (DR); Cataracts; and Stargardt Disease (SD)

It has long been appreciated that oxidative stress plays a major role inmany retinal degeneration conditions (Winkler B S et al, MolecularVision 1999; 5:32). Increased oxygen levels, exposure to light and highPUFA content lead to increased PUFA peroxidation in the eye tissues.AMD, the leading cause of blindness in elderly populations in thedeveloped world, is accompanied by degeneration of retinal pigmentepithelial cells (RPE). Although smoking, atherosclerosis, geneticfactors and fibrinogen have been implicated in the pathogenesis of thisdisease, the exact cause of AMD remains unknown (Baskol G. Et al.Ophthalmol. 2006; 220:12-16). Oxidative stress plays a major role in thepathogenesis of AMD (Beatty S, et al. Survey Ophtalm. 2000; 45:115-134).The retinal pigment epithelium (RPE) is essential for retinoid recyclingand phagocytosis of photoreceptors. Methods of comparative proteomicshave been applied to uncover global changes of RPE proteins that mediateoxidative stress-induced degeneration of RPE (Arnouk H et al, J.Proteom. 2011; 74:254-261). Increased levels of PUFA peroxidationproducts such as HNE and HHE (Long E K, et al. Free Rad. Biol. Med.2010; 49:1-8) as well as MDA (Singh S B et al, J. Clin. Diagnostic Res.2010; 4:2768-2769) have been reported in retinas, while increased levelof oxidized phospholipids in photoreceptors and RPE is associated withaging and AMD (Suzuki M et al, Molec. Vision 2007; 13:772-778). PUFAperoxidation products play a major role in formation of retinal pigmentepithelial (RPE) lipofuscin, which itself, and mostly through its A2Ecomponent, can generate ROS upon irradiation with visible light andplays a major role in etiology of AMD (Katz M L, Arch. Gerontol.Geriatr. 2002; 34:359-370). In Stargardt disease (SD), lipofuscincontent in the photoreceptor outer segment and RPE is increased due toABCA4 mutations (Weng J et al, PNAS 2000; 97:7154-7159). PUFAperoxidation products, including MDA, play such a prominent role in lenspathologies including formation of cataracts, that the PUFA peroxidationwas proclaimed to be an initiating step in the human cataractpathogenesis (Borchman D. et al, J. Lipid Res. 2010; 51:2473-2488).Equally important is the role of PUFA peroxidation products inpathophysiology of diseases of human cornea, including pterygium andkeratoconus (Shoham A, et al. Free Rad. Biol. Med. 2008; 45:1047-1055).Diabetic retinopathy is also associated with oxidative stress and PUFAperoxidation (Baynes J W, Thorpe S R. Diabetes 1999; 48:1-9).Peroxidation of linoleic acid and other PUFAs that make up LDL has beenshown to affect exudative AMD (Javadzadeh a et al, Molec. Vision 2010;16:275-276).

Despite the important role the oxidative stress plays in thesedegenerative conditions, the success of the antioxidant therapies has sofar been limited. This may be due to several reasons: (a) antioxidantsare usually present in cells at high (virtually saturated)concentrations, and further supplementation leads to only marginalincreases (Zimniak P Ageing Res. Rev. 2008; 7:281-300). The stochasticnature of ROS-inflicted damage is therefore not sensitive to antioxidanttherapies; (b) ROS themselves are important in cell signaling and otherprocesses, including the requirement for low levels of ROS for hormetic(adaptive) upregulation of protective mechanisms; (c) some antioxidants(such as vitamin E) can become potent oxidants themselves, capable ofinitiating PUFA autoxidation (Bowry V W et al, JACS 1993;115:6029-6044). Moreover, vitamin A plays a major role in formation ofRPE-associated lipofuscin fluorophore A2E and its derivatives, which arecapable of generating ROS upon exposure to irradiation (Sparrow J R etal, Investigative Ophthalm. Vis. Sci. 1999; 40:2988-2995); and (d)antioxidants are ineffective in neutralising the carbonyl compounds likeFINE and HHE, because FINE and HHE, once formed, react in different wayscompared to the free radical mechanism and so cannot be quenched bytypical antioxidants.

In some aspects, identification of a subject who has or is susceptibleto AMD, RP, DR, cataracts and/or SD may be determined by diagnostictests known in the art such as fluorescein angiography or by identifyingabnormalities in vascular processes. In addition, Optical CoherenceTomography diagnostics may be used to identify such subjects.

Some aspects of this invention arise from: (1) an understanding thatwhile essential PUFAs are vital for proper functioning of lipidmembranes, and in particular of the mitochondrial membranes, theirinherent drawback, i.e., the propensity to be oxidized by ROS withdetrimental outcome, is implicated in Wet and Dry Age-related MacularDegeneration (AMD); Retinitis Pigmentosa (RP); Diabetic Retinopathy(DR); Cataracts; and Stargardt Disease (SD); (2) antioxidants cannotprevent PUFA peroxidation due to stochastic nature of the process andthe stability of PUFA peroxidation products (reactive carbonyls) toantioxidant treatment, and (3) the ROS-driven damage of oxidation-pronesites within PUFAs may be overcome by using an approach that makes themless amenable to such oxidations, without compromising any of theirbeneficial physical properties. Some aspects of this invention describethe use of the isotope effect to achieve this, only at sites inessential PUFAs and PUFA precursors that matter most for oxidation,while other aspects contemplate other sites in addition to those thatmatter most for oxidation.

Isotopically labeled embodiments should have minimal or non-existenteffects on important biological processes. For example, the naturalabundance of isotopes present in biological substrates implies that lowlevels of isotopically labeled compounds should have negligible effectson biological processes. Additionally, hydrogen atoms are incorporatedinto biological substrates from water, and is it known that theconsumption of D₂O, or heavy water, does not pose a health threat tohumans. See, e.g., “Physiological effect of heavy water.” Elements andisotopes: formation, transformation, distribution. Dordrecht: KluwerAcad. Publ. (2003) pp. 111-112 (indicating that a 70 kg person mightdrink 4.8 liters of heavy water without serious consequences). Moreover,many isotopically labeled compounds are approved by the U.S. Food & DrugAdministration for diagnostic and treatment purposes.

It will be appreciated by those skilful in the art that the same effectcan be achieved by protecting oxidation-prone positions within PUFAsusing other chemical approaches. Certain PUFA mimetics, while possessingstructural similarity with natural PUFAs, will nevertheless be stable toROS-driven oxidation due to structural reinforcement.

Compositions:

In some embodiments, an isotopically modified polyunsaturated fatty acidor a mimetic refers to a compound having structural similarity to anaturally occurring PUFA that is stabilized chemically or byreinforcement with one or more isotopes, for example ¹³C and/ordeuterium. Generally, if deuterium is used for reinforcement, one orboth hydrogens on a methylene group may be reinforced.

Some aspects of this invention provide compounds that are analogues ofessential PUFAs with either one, several, or all bis-allylic positionssubstituted with heavy isotopes. In some embodiments, the CH₂ groups,which will become the bis-allylic position in a PUFA upon enzymaticconversion, are substituted with one or two heavy isotopes. Suchcompounds are useful for the prevention or treatment of diseases inwhich PUFA oxidation is a factor or can contribute to diseaseprogression.

The bis-allylic position generally refers to the position of thepolyunsaturated fatty acid or mimetic thereof that corresponds to themethylene groups of 1,4-diene systems. The pro-bis-allylic positionrefers to the methylene group that becomes the bis-allylic position uponenzymatic desaturation.

In some embodiments, the chemical identity of PUFAs, i.e., the chemicalstructure without regard to the isotope substitutions or substitutionsthat mimic isotope substitutions, remains the same upon ingestion. Forinstance, the chemical identity of essential PUFAs, that is, PUFAs thatmammals such as humans do not generally synthesize, may remain identicalupon ingestion. In some cases, however, PUFAs may be furtherextended/desaturated in mammals, thus changing their chemical identityupon ingestion. Similarly with mimetics, the chemical identity mayremain unchanged or may be subject to similar extension/desaturation. Insome embodiments, PUFAs that are extended, and optionally desaturated,upon ingestion and further metabolism may be referred to as higherhomologs.

In some embodiments, naturally-occurring abundance level refers to thelevel of isotopes, for example ¹³C and/or deuterium that may beincorporated into PUFAs that would be relative to the natural abundanceof the isotope in nature. For example, ¹³C has a natural abundance ofroughly 1% ¹³C atoms in total carbon atoms. Thus, the relativepercentage of carbon having greater than the natural abundance of ¹³C inPUFAs may have greater than the natural abundance level of roughly 1% ofits total carbon atoms reinforced with ¹³C, such as 2%, but preferablyabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% of ¹³C with respect to one or morecarbon atoms in each PUFA molecule. In other embodiments, the percentageof total carbon atoms reinforced with ¹³C is at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100%.

Regarding hydrogen, in some embodiments, deuterium has a naturalabundance of roughly 0.0156% of all naturally occurring hydrogen in theoceans on earth. Thus, a PUFA having greater that the natural abundanceof deuterium may have greater than this level or greater than thenatural abundance level of roughly 0.0156% of its hydrogen atomsreinforced with deuterium, such as 0.02%, but preferably about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% of deuterium with respect to one or more hydrogenatoms in each PUFA molecule. In other embodiments, the percentage oftotal hydrogen atoms reinforced with deuterium is at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%.

In some aspects, a composition of PUFAs contains both isotopicallymodified PUFAs and isotopically unmodified PUFAs. The isotopic purity isa comparison between a) the relative number of molecules of isotopicallymodified PUFAs, and b) the total molecules of both isotopically modifiedPUFAs and PUFAs with no heavy atoms. In some embodiments, the isotopicpurity refers to PUFAs that are otherwise the same except for the heavyatoms.

In some embodiments, isotopic purity refers to the percentage ofmolecules of an isotopically modified PUFAs in the composition relativeto the total number of molecules of the isotopically modified PUFAs plusPUFAs with no heavy atoms. For example, the isotopic purity may be about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of the molecules of isotopicallymodified PUFAs relative to the total number of molecules of both theisotopically modified PUFAs plus PUFAs with no heavy atoms. In otherembodiments, the isotopic purity is at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%. In some embodiments, isotopic purity of the PUFAs may be fromabout 10%-100%, 10%-95%, 10%-90%, 10%-85%, 10%-80%, 10%-75%, 10%-70%,10%-65%, 10%-60%, 10%-55%, 10%-50%, 10%-45%, 10%-40%, 10%-35%, 10%-30%,10%-25%, or 10%-20% of the total number of molecules of the PUFAs in thecomposition. In other embodiments, isotopic purity of the PUFAs may befrom about 15%-100%, 15%-95%, 15%-90%, 15%-85%, 15%-80%, 15%-75%,15%-70%, 15%-65%, 15%-60%, 15%-55%, 15%-50%, 15%-45%, 15%-40%, 15%-35%,15%-30%, 15%-25%, or 15%-20% of the total number of molecules of thePUFAs in the composition. In some embodiments, isotopic purity of thePUFAs may be from about 20%-100%, 20%-95%, 20%-90%, 20%-85%, 20%-80%,20%-75%, 20%-70%, 20%-65%, 20%-60%, 20%-55%, 20%-50%, 20%-45%, 20%-40%,20%-35%, 20%-30%, or 20%-25% of the total number of molecules of thePUFAs in the composition. Two molecules of an isotopically modified PUFAout of a total of 100 total molecules of isotopically modified PUFAsplus PUFAs with no heavy atoms will have 2% isotopic purity, regardlessof the number of heavy atoms the two isotopically modified moleculescontain.

In some aspects, an isotopically modified PUFA molecule may contain onedeuterium atom, such as when one of the two hydrogens in a methylenegroup is replaced by deuterium, and thus may be referred to as a “D1”PUFA. Similarly, an isotopically modified PUFA molecule may contain twodeuterium atoms, such as when the two hydrogens in a methylene group areboth replaced by deuterium, and thus may be referred to as a “D2” PUFA.Similarly, an isotopically modified PUFA molecule may contain threedeuterium atoms and may be referred to as a “D3” PUFA. Similarly, anisotopically modified PUFA molecule may contain four deuterium atoms andmay be referred to as a “D4” PUFA. In some embodiments, an isotopicallymodified PUFA molecule may contain five deuterium atoms or six deuteriumatoms and may be referred to as a “D5” or “D6” PUFA, respectively.

The number of heavy atoms in a molecule, or the isotopic load, may vary.For example, a molecule with a relatively low isotopic load may containabout 1, 2, 3, 4, 5, or 6 deuterium atoms. A molecule with a moderateisotopic load may contain about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 deuterium atoms. In a molecule with a very high load, everyhydrogen may be replaced with a deuterium. Thus, the isotopic loadrefers to the percentage of heavy atoms in each PUFA molecule. Forexample, the isotopic load may be about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of the number of the same type of atoms in comparison to a PUFA with noheavy atoms of the same type (e.g. hydrogen would be the “same type” asdeuterium). In some embodiments, the isotopic load is at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%. Unintended side effects are expected to bereduced where there is high isotopic purity in a PUFA composition butlow isotopic load in a given molecule. For example, the metabolicpathways will likely be less affected by using a PUFA composition withhigh isotopic purity but low isotopic load.

One will readily appreciate that when one of the two hydrogens of amethylene group is replaced with a deuterium atom, the resultantcompound may possess a stereocenter. In some embodiments, it may bedesirable to use racemic compounds. In other embodiments, it may bedesirable to use enantiomerically pure compounds. In additionalembodiments, it may be desirable to use diastereomerically purecompounds. In some embodiments, it may be desirable to use mixtures ofcompounds having enantiomeric excesses and/or diastereomeric excesses ofabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100%. In other embodiments, theenantiomeric excesses and/or diastereomeric excesses is at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100%. In some embodiments, it may be preferableto utilize stereochemically pure enantiomers and/or diastereomers ofembodiments—such as when contact with chiral molecules is being targetedfor attenuating oxidative damage. However, in many circumstances,non-chiral molecules are being targeted for attenuating oxidativedamage. In such circumstances, embodiments may be utilized withoutconcern for their stereochemical purity. Moreover, in some embodiments,mixtures of enantiomers and diastereomers may be used even when thecompounds are targeting chiral molecules for attenuating oxidativedamage.

In some aspects, isotopically modified PUFAs impart an amount of heavyatoms in a particular tissue. Thus, in some aspects, the amount of heavymolecules will be a particular percentage of the same type of moleculesin a tissue. For example, the number of heavy molecules may be about1%-100% of the total amount of the same type of molecules. In someaspects, 10-50% the molecules are substituted with the same type ofheavy molecules.

In some embodiments, a compound with the same chemical bonding structureas an essential PUFA but with a different isotopic composition atparticular positions will have significantly and usefully differentchemical properties from the unsubstituted compound. The particularpositions with respect to oxidation, including oxidation by ROS,comprise bis-allylic positions of essential polyunsaturated fatty acidsand their derivatives, as shown in FIG. 1. The essential PUFAsisotopically reinforced at bis-allylic positions shown below will bemore stable to the oxidation. Accordingly, some aspects of the inventionprovide for particular methods of using compounds of Formula (1) orsalts thereof, whereas the sites can be further reinforced withcarbon-13. R¹=alkyl, H, or cation; m=1-10; n=1-5, where at eachbis-allylic position, one or both Y atoms are deuterium atoms, forexample,

11,11-Dideutero-cis,cis-9,12-Octadecadienoic acid(11,11-Dideutero-(9Z,12Z)-9,12-Octadecadienoic acid; D2-LA); and11,11,14,14-Tetradeutero-cis,cis,cis-9,12,15-Octadecatrienoic acid(11,11,14,14-Tetradeutero-(9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid;D4-ALA). In some embodiments, said positions, in addition todeuteration, can be further reinforced by carbon-13, each at levels ofisotope abundance above the naturally-occurring abundance level. Allother carbon-hydrogen bonds in the PUFA molecule may optionally containdeuterium and/or carbon-13 at, or above, the natural abundance level.

Essential PUFAs are biochemically converted into higher homologues bydesaturation and elongation. Therefore, some sites which are notbis-allylic in the precursor PUFAs will become bis-allylic uponbiochemical transformation. Such sites then become sensitive tooxidation, including oxidation by ROS. In a further embodiment, suchpro-bis-allylic sites, in addition to existing bis-allylic sites arereinforced by isotope substitution as shown below. Accordingly, thisaspect of the invention provides for the use of compounds of Formula (2)or salt thereof, where at each bis-allylic position, and at eachpro-bis-allylic position, one or more of X or Y atoms may be deuteriumatoms. R1=alkyl, cation, or H; m=1-10; n=1-5; p=1-10.

Said positions, in addition to deuteration, can be further reinforced bycarbon-13, each at levels of isotope abundance above thenaturally-occurring abundance level. All other carbon-hydrogen bonds inthe PUFA molecule may contain optionally deuterium and/or carbon-13 ator above the natural abundance level.

Oxidation of PUFAs at different bis-allylic sites gives rise todifferent sets of oxidation products. For example, 4-HNE is formed fromn-6 PUFAs whereas 4-HHE is formed from n-3 PUFAs (Negre-Salvayre A, etal. Brit. J. Pharmacol. 2008; 153:6-20). The products of such oxidationpossess different regulatory, toxic, signaling, etc. properties. It istherefore desirable to control the relative extent of such oxidations.Accordingly, some aspects of the invention provide for the use ofcompounds of Formula (3), or salt thereof, differentially reinforcedwith heavy stable isotopes at selected bis-allylic or pro-bis-allylicpositions, to control the relative yield of oxidation at differentsites, as shown below, such that any of the pairs of Y¹-Y^(n) and/orX¹-X^(m) at the bis-allylic or pro-bis-allylic positions of PUFAs maycontain deuterium atoms. R1=alkyl, cation, or H; m=1-10; n=1-6; p=1-10

Said positions, in addition to deuteration, can be further reinforced bycarbon-13. All other carbon-hydrogen bonds in the PUFA molecule maycontain deuterium at, or above the natural abundance level. It will beappreciated that the break lines in the structure shown above representsa PUFA with a varying number of double bonds, a varying number of totalcarbons, and a varying combination of isotope reinforced bis-allylic andpro-bis-allylic sites.

Exact structures of compounds illustrated above are shown below thatprovide for both isotope reinforced n-3 (omega-3) and n-6 (omega-6)essential polyunsaturated fatty acids, and the PUFAs made from thembiochemically by desaturation/elongation. Any one of these compounds maybe used to slow oxidation. In the following compounds, the PUFAs areisotopically reinforced at oxidation sensitive sites and/or sites thatmay become oxidation sensitive upon biochemical desaturation/elongation.R¹ may be H, alkyl, or cation; R² may be H or D; * represents either ¹²Cor ¹³C.

D-Linoleic acids include:

The per-deuterated linoleic acid below may be produced bymicrobiological methods, for example by growing in media containingdeuterium and/or carbon-13.

D-Arachidonic acids include:

The per-deuterated arachidonic acid below may be produced bymicrobiological methods, such as by growing in media containingdeuterium and/or carbon-13.

D-Linolenic acids include:

Per-deuterated linolenic acid below may be produced by microbiologicalmethods, such as growing in media containing deuterium and/or carbon-13.

In some aspects of the invention, any PUFAs, whether essential or not,that are capable of being taken up from diet and used in the body, canbe utilized. In the case of essential or non-essential PUFAs orprecursors, the supplemented stabilized materials can compete with otherdietary uptake and bio-manufacture to reduce the availabledisease-causing species concentrations.

In some aspects of the invention, the PUFAs isotopically reinforced atoxidation sensitive positions as described by way of the structuresabove are heavy isotope enriched at said positions as compared to thenatural abundance of the appropriate isotope, deuterium and/orcarbon-13.

In some embodiments, the disclosed compounds are enriched to 99% isotopepurity or more. In some embodiments, the heavy isotope enrichment atsaid positions is between 50%-99% deuterium and/or carbon-13.

In some embodiments, the modified fatty acids, when dosed via diet asdrugs or supplements, may be dosed as prodrugs, including, but notlimited to, non-toxic and pharmaceutically suitable esters of the parentfatty acid or mimetic, such as an ethyl ester or glyceryl ester. Thisester assists in tolerance of the drug in the gut, assists in digestion,and relies on the high levels of esterases in the intestines tode-esterify the ester pro-drugs into the active acid form of the drugwhich adsorbs. Hence, in some embodiments, the invention encompasses thepro-drug esters of the modified fatty acids herein. Examples of thistype of drug in the market, nutrition, and clinical trials literature,including Glaxo's Lovaza, (mixtures of omega 3 fatty acid esters, EPA,DHA, and alpha-linolenic acid), Abbott's Omacor (omega-3-fatty acidesters), and most fish oil supplements (DHA and EPA esters). In someaspects, incorporation of the ester pro-drugs into tissues or cellsrefers to the incorporation of the modified parent PUFA as it would beused as a bodily constituent.

In some embodiments, stabilized compositions mimic natural occurringfatty acids without changing their elemental composition. For example,the substituent may retain the chemical valence shell. Some embodimentsinclude naturally occurring fatty acids, mimetics, and their esterpro-drugs, that are modified chemically to be effective at preventingspecific disease mechanisms, but are modified in a way (such as isotopicsubstitution) that does not change the elemental composition of thematerial. For example, deuterium is a form of the same element hydrogen.In some aspects, these compounds maintain elemental composition and arestabilized against oxidation. Some compounds that are stabilized againstoxidation are stabilized at oxidation sensitive loci. Some compounds arestabilized against oxidation via heavy isotope substitution, then atbis-allylic carbon hydrogen bonds, etc.

In a further embodiment, oxidation-prone bis-allylic sites of PUFAs canbe protected against hydrogen abstraction by moving bis-allylichydrogen-activating double bonds further apart, thus eliminating thebis-allylic positions while retaining certain PUFA fluidity as shownbelow. These PUFA mimetics have no bis-allylic positions.

In a further embodiment, oxidation-prone bis-allylic sites of PUFAs canbe protected against hydrogen abstraction by using heteroatoms withvalence II, thus eliminating the bis-allylic hydrogens as shown below.These PUFA mimetics also have no bis-allylic hydrogens.

In a further embodiment, PUFA mimetics, i.e. compounds structurallysimilar to natural PUFAs but unable to get oxidized because of thestructural differences, can be employed for the above mentionedpurposes. Oxidation-prone bis-allylic sites of PUFAs can be protectedagainst hydrogen abstraction by di-methylation or halogenation as shownbelow. The hydrogen atoms on the methyl groups may optionally behalogens, such as fluorine, or deuterium. These PUFA mimetics aredimethylated at bis-allylic sites.

In a further embodiment, oxidation-prone bis-allylic sites of PUFAs canbe protected against hydrogen abstraction by alkylation as shown below.These PUFA mimetics are dialkylated at bis-allylic sites.

In a further embodiment, cyclopropyl groups can be used instead ofdouble bonds, thus rendering the acids certain fluidity whileeliminating the bis-allylic sites as shown below. These PUFA mimeticshave cyclopropyl groups instead of double bonds.

In a further embodiment, 1,2-substituted cyclobutyl groups inappropriate conformation can be used instead of double bonds, thusrendering the acids certain fluidity while eliminating the bis-allylicsites as shown below. These PUFA mimetics have 1,2-cyclobutyl groupsinstead of double bonds.

In a modification of the previous embodiment of mimetics with1,2-cyclobutyl groups instead of double bonds, 1,3-substitutedcyclobutyl groups in appropriate conformation can be used instead ofdouble bonds, thus rendering the acids certain fluidity whileeliminating the bis-allylic sites. The following PUFA mimetics have1,3-cyclobutyl groups instead of double bonds.

It is a well known principle in medicinal chemistry that certainfunctional groups are isosteric and/or bioisosteric with certain otherfunctional groups. Bioisosteres are substituents or groups with similarphysical or chemical properties which produce broadly similar biologicalproperties to a chemical compound. For example, well known isosteresand/or bioisosteres for hydrogen include halogens such as fluorine;isosteres and/or bioisosteres of alkenes include alkynes, phenyl rings,cyclopropyl rings, cyclobutyl rings, cyclopentyl rings, cyclohexylrings, thioethers, and the like; isosteres and/or bioisosteres ofcarbonyls include sulfoxides, sulfones, thiocarbonyls, and the like;isosteres and/or bioisosteres of esters include amides, sulfonic acidesters, sulfonamides, sulfinyl acid esters, sulfinylamindes, and thelike. Consequently, PUFA mimetics also include compounds havingisosteric and/or bioisosteric functional groups.

It is contemplated that it may be useful to formulate PUFAs and/or PUFAmimetics as a pro-drug for use in the invention. A pro-drug is apharmacological substance may itself have biological activity, but uponadministration the pro-drug is metabolized into a form that also exertsbiological activity. Many different types of pro-drugs are known andthey can be classified into two major types based upon their cellularsites of metabolism. Type I pro-drugs are those that are metabolizedintracellularly, while Type II are those that are metabolizedextracellularly. It is well-known that carboxylic acids may be convertedto esters and various other functional groups to enhancepharmacokinetics such as absorption, distribution, metabolism, andexcretion. Esters are a well-known pro-drug form of carboxylic acidsformed by the condensation of an alcohol (or its chemical equivalent)with a carboxylic acid (or its chemical equivalent). In someembodiments, alcohols (or their chemical equivalent) for incorporationinto pro-drugs of PUFAs include pharmaceutically acceptable alcohols orchemicals that upon metabolism yield pharmaceutically acceptablealcohols. Such alcohols include, but are not limited to, propyleneglycol, ethanol, isopropanol, 2-(2-ethoxyethoxy)ethanol (Transcutol®,Gattefosse, Westwood, N.J. 07675), benzyl alcohol, glycerol,polyethylene glycol 200, polyethylene glycol 300, or polyethylene glycol400; polyoxyethylene castor oil derivatives (for example,polyoxyethyleneglyceroltriricinoleate or polyoxyl 35 castor oil(Cremophor®EL, BASF Corp.), polyoxyethyleneglycerol oxystearate(Cremophor®RH 40 (polyethyleneglycol 40 hydrogenated castor oil) orCremophor®RH 60 (polyethyleneglycol 60 hydrogenated castor oil), BASFCorp.)); saturated polyglycolized glycerides (for example, Gelucire®35/10, Gelucire® 44/14, Gelucire® 46/07, Gelucire® 50/13 or Gelucire®53/10, available from Gattefosse, Westwood, N.J. 07675); polyoxyethylenealkyl ethers (for example, cetomacrogol 1000); polyoxyethylene stearates(for example, PEG-6 stearate, PEG-8 stearate, polyoxyl 40 stearate NF,polyoxyethyl 50 stearate NF, PEG-12 stearate, PEG-20 stearate, PEG-100stearate, PEG-12 distearate, PEG-32 distearate, or PEG-150 distearate);ethyl oleate, isopropyl palmitate, isopropyl myristate; dimethylisosorbide; N-methylpyrrolidinone; parafin; cholesterol; lecithin;suppository bases; pharmaceutically acceptable waxes (for example,carnauba wax, yellow wax, white wax, microcrystalline wax, oremulsifying wax); pharmaceutically acceptable silicon fluids; soribitanfatty acid esters (including sorbitan laurate, sorbitan oleate, sorbitanpalmitate, or sorbitan stearate); pharmaceutically acceptable saturatedfats or pharmaceutically acceptable saturated oils (for example,hydrogenated castor oil (glyceryl-tris-12-hydroxystearate), cetyl esterswax (a mixture of primarily C14-C18 saturated esters of C14-C18saturated fatty acids having a melting range of about 43°-47° C.), orglyceryl monostearate).

In some embodiments, the fatty acid pro-drug is represented by the esterP-B, wherein the radical P is a PUFA and the radical B is a biologicallyacceptable molecule. Thus, cleavage of the ester P-B affords a PUFA anda biologically acceptable molecule. Such cleavage may be induced byacids, bases, oxidizing agents, and/or reducing agents. Examples ofbiologically acceptable molecules include, but are not limited to,nutritional materials, peptides, amino acids, proteins, carbohydrates(including monosaccharides, disaccharides, polysaccharides,glycosaminoglycans, and oligosaccharides), nucleotides, nucleosides,lipids (including mono-, di- and tri-substituted glycerols,glycerophospholipids, sphingolipids, and steroids).

In some embodiments, alcohols (or their chemical equivalent) forincorporation into pro-drugs of PUFAs include alcohols with 1 to 50carbon atoms (“C₁₋₅₀ alcohols”), C₁₋₄₅ alcohols, C₁₋₄₀ alcohols, C₁₋₃₅alcohols, C₁₋₃₀ alcohols, C₁₋₂₅ alcohols, C_(1-20 alcohols, C) ₁₋₁₅alcohols, C₁₋₁₀ alcohols, C₁₋₆ alcohols (whenever it appears herein, anumerical range such as “1-50” refers to each integer in the givenrange; e.g., “1 to 50 carbon atoms” means that the alkyl group mayconsist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up toand including 50 carbon atoms, although the present definition alsocovers the occurrence of the term “alkyl” where no numerical range isdesignated). Such alcohols may be branched, unbranched, saturated,unsaturated, polyunsaturated and/or include one or more heteroatoms suchas nitrogen, oxygen, sulfur, phosphorus, boron, silicone, fluorine,chlorine, bromine, or iodine. Exemplary alcohols include methyl, ethyl,propyl, iso-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl,hexyl, perfluoromethyl, perchloromethyl, perfluoro-tert-butyl,perchloro-tert-butyl, and benzyl alcohols as well as ether alcohols suchas polyethylene glycols. In some embodiments, the alcohol contains acharged species. Such species may be anionic or cationic. In someembodiments, the species is a positively charged phosphorus atom. Inother embodiments, the positively charged phosphorus atom is aphosphonium cation. In other embodiments the charged species is aprimary, secondary, tertiary, or quaternary ammonium cation.

In some embodiments, alcohols (or their chemical equivalent) forincorporation into pro-drugs of PUFAs include polyalcohols such asdiols, triols, tetra-ols, penta-ols, etc. Examples of polyalcoholsinclude ethylene glycol, propylene glycol, 1,3-butylene glycol,polyethylene glycol, methylpropanediol, ethoxydiglycol, hexylene glycol,dipropylene glycol glycerol, and carbohydrates. Esters formed frompolyalcohols and PUFAs may be mono-esters, di-esters, tri-esters, etc.In some embodiments, multiply esterified polyalcohols are esterifiedwith the same PUFAs. In other embodiments, multiply esterifiedpolyalcohols are esterified with different PUFAs. In some embodiments,the different PUFAs are stabilized in the same manner. In otherembodiments, the different PUFAs are stabilized in different manners(such as deuterium substitution in one PUFA and ¹³C substitution inanother PUFA). In some embodiments, one or more PUFAs is an omega-3fatty acid and one or more PUFAs is an omega-6 fatty acid.

It is also contemplated that it may be useful to formulate PUFAs and/orPUFA mimetics and/or PUFA pro-drugs as salts for use in the invention.For example, the use of salt formation as a means of tailoring theproperties of pharmaceutical compounds is well known. See Stahl et al.,Handbook of pharmaceutical salts: Properties, selection and use (2002)Weinheim/Zurich: Wiley-VCH/VHCA; Gould, Salt selection for basic drugs,Int. J. Pharm. (1986), 33:201-217. Salt formation can be used toincrease or decrease solubility, to improve stability or toxicity, andto reduce hygroscopicity of a drug product.

Formulation of PUFAs and/or PUFA mimetics and/or PUFA pro-drugs as saltsincludes, but is not limited to, the use of basic inorganic salt formingagents, basic organic salt forming agents, and salt forming agentscontaining both acidic and basic functional groups. Various usefulinorganic bases for forming salts include, but are not limited to,alkali metal salts such as salts of lithium, sodium, potassium rubidium,cesium, and francium, and alkaline earth metal salts such as berylium,magnesium, calcium, strontium, barium, and radium, and metals such asaluminum. These inorganic bases may further include counterions such ascarbonates, hydrogen carbonates, sulfates, hydrogen sulfates, sulfites,hydrogen sulfites, phosphates, hydrogen phosphates, dihydrogenphosphates, phosphites, hydrogen phosphites, hydroxides, oxides,sulfides, alkoxides such as methoxide, ethoxide, and t-butoxide, and thelike. Various useful organic bases for forming salts include, but arenot limited to, amino acids, basic amino acids such as arginine, lysine,ornithine and the like, ammonia, alkylamines such as methylamine,ethylamine, dimethylamine, diethylamine, trimethylamine, triethylamineand the like, heterocyclic amines such as pyridine, picoline and thelike, alkanolamines such as ethanolamine, diethanolamine,triethanolamine and the like, diethylaminoethanol, dimethylaminoethanol,N-methylglucamine, dicyclohexylamine, N,N′-dibenzylethylenediamine,ethylenediamine, piperazine, choline, trolamine, imidazole, diolamine,betaine, tromethamine, meglumine, chloroprocain, procaine, and the like.

Salt formulations of PUFAs and/or PUFA mimetics and/or PUFA pro-drugsinclude, but are not limited to, pharmaceutically acceptable basicinorganic salts, basic organic salts, and/or organic compounds havingboth acidic and basic functional groups. Pharmaceutically acceptablesalts are well known in the art and include many of the above-recitedinorganic and organic bases. Pharmaceutically acceptable salts furtherinclude salts and salt-forming agents found in drugs approved by theFood and Drug Administration and foreign regulatory agencies.Pharmaceutically acceptable organic cations for incorporation include,but are not limited to, benzathine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine, procaine, benethamine,clemizole, diethylamine, piperazine, and tromethamine. Pharmaceuticallyacceptable metallic cations for incorporation include, but are notlimited to, aluminum, calcium, lithium, magnesium, potassium, sodium,zinc, barium, and bismuth. Additional salt-forming agents include, butare not limited to, arginine, betaine, carnitine, diethylamine,L-glutamine, 2-(4-imidazolyl)ethylamine, isobutanolamine, lysine,N-methylpiperazine, morpholine, and theobromine.

Moreover, several lists of pharmaceutically approved counterions exists.See Bighley et al., Salt forms of drugs and absorption. 1996 In:Swarbrick J. et al. eds. Encyclopaedia of pharmaceutical technology,Vol. 13 New York: Marcel Dekker, Inc. pp 453-499; Gould, P. L., Int. J.Pharm. 1986, 33, 201-217; Berge, J. Pharm. Sci. 1977, 66, 1-19; HeinrichStahl P., Wermuch C. G. (editors), Handbook of Pharmaceutical Salts,IUPAC, 2002; Stahl et al., Handbook of pharmaceutical salts: Properties,selection and use (2002) Weinheim/Zurich: Wiley-VCH/VHCA, all of whichare incorporated herein by reference.

It may be unnecessary to substitute all isotopically unmodified PUFAs,such as non-deuterated PUFAs, with isotopically modified PUFAs such asdeuterated PUFAs. In some embodiments, is preferable to have sufficientisotopically modified PUFAs such as D-PUFAs in the membrane to preventunmodified PUFAs such as H-PUFAs from sustaining a chain reaction ofself-oxidation. During self-oxidation, when one PUFA oxidizes, and thereis a non-oxidized PUFA in the vicinity, the non-oxidized PUFA can getoxidized by the oxidized PUFA. This may also be referred to asautooxidation. In some instances, if there is a low concentration, forexample “dilute” H-PUFAs in the membrane with D-PUFAs, this oxidationcycle may be broken due to the distance separating H-PUFAs. In someembodiments, the concentration of isotopically modified PUFAs is presentin a sufficient amount to maintain autooxidation chain reaction. Tobreak the autooxidation chain reaction, for example, 1-60%, 5-50%, or15-35% of the total molecules of the same type are in the membrane. Thismay be measured by IRMS (isotope ratio mass spectrometry).

A further aspect of the invention provides a dietary, supplementary orpharmaceutical composition of the active compounds. In some embodiments,the dietary, supplementary, or pharmaceutical composition may comprise asalt of the active compound.

Various useful inorganic bases for forming salts include, but are notlimited to, alkali metal salts such as salts of lithium, sodium,potassium rubidium, cesium, and francium, and alkaline earth metal saltssuch as berylium, magnesium, calcium, strontium, barium, and radium, andmetals such as aluminum. These inorganic bases may further includecounterions such as carbonates, hydrogen carbonates, sulfates, hydrogensulfates, sulfites, hydrogen sulfites, phosphates, hydrogen phosphates,dihydrogen phosphates, phosphites, hydrogen phosphites, hydroxides,oxides, sulfides, alkoxides such as methoxide, ethoxide, and t-butoxide,and the like.

Various useful organic bases for forming salts include, but are notlimited to, amino acids; basic amino acids such as arginine, lysine,ornithine and the like; ammonia; ammonium hydroxide; alkylamines such asmethylamine, ethylamine, dimethylamine, diethylamine, trimethylamine,triethylamine and the like; heterocyclic amines such as pyridine,picoline and the like; alkanolamines such as ethanolamine,diethanolamine, triethanolamine and the like, diethylaminoethanol,dimethylaminoethanol; N-methylglucamine; dicyclohexylamine;N,N′-dibenzylethylenediamine; ethylenediamine; piperazine; choline;trolamine; imidazole; diolamine; betaine; tromethamine; meglumine;chloroprocain; procaine; and the like.

Salts of active compounds may include, but are not limited to,pharmaceutically acceptable salts. Pharmaceutically acceptable salts arewell known in the art and include many of the above-listed salt-formingagents. Pharmaceutically acceptable salts further include salts andsalt-forming agents of the type present in drugs approved by the Foodand Drug Administration and foreign regulatory agencies.

Pharmaceutically acceptable organic cations for incorporation into asalt of an active compound include, but are not limited to, benzathine,chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine,procaine, benethamine, clemizole, diethylamine, piperazine, andtromethamine.

Pharmaceutically acceptable metallic cations for incorporation into asalt of an active compound include, but are not limited to, aluminum,calcium, lithium, magnesium, potassium, sodium, zinc, barium, andbismuth.

Additional salt-forming agents having potential usefulness as formingsalts include, but are not limited to, acetylaminoacetic acid,N-acetyl-L-asparagine, N-acetylcystine, arginine, betaine, carnitine,L-glutamine, 2-(4-imidazolyl)ethylamine, isobutanolamine, lysine,N-methylpiperazine, and morpholine.

Moreover, several lists of pharmaceutically approved counterions exists.See Bighley et al., Salt forms of drugs and absorption. 1996 In:Swarbrick J. et al. eds. Encyclopaedia of pharmaceutical technology,Vol. 13 New York: Marcel Dekker, Inc. pp 453-499; Gould, P. L., Int. J.Pharm. 1986, 33, 201-217; Berge, J. Pharm. Sci. 1977, 66, 1-19; HeinrichStahl P., Wermuch C. G. (editors), Handbook of Pharmaceutical Salts,IUPAC, 2002; Stahl et al., Handbook of pharmaceutical salts: Properties,selection and use (2002) Weinheim/Zurich: Wiley-VCH/VHCA, all of whichare incorporated herein by reference.

Co-Administration

In some embodiments, the compounds disclosed herein are administered incombination. For example, in some embodiments, two, three, four, and/orfive or more stabilized compounds are administered together. In someembodiments, stabilized compounds are administered in approximatelysimilar amounts. In other embodiments, stabilized compounds areadministered in differing amounts. For example, any one of two or morecompounds in a mixture may represent about 1% to about 99% of a mixture,about 5% to about 95% of a mixture, about 10% to about 90% of a mixture,about 15% to about 85% of a mixture, about 20% to about 80% of amixture, about 25% to about 75% of a mixture, about 30% to about 70% ofa mixture, about 35% to about 65% of a mixture, about 40% to about 60%of a mixture, about 40% to about 60% of a mixture, about 45% to about55% of a mixture, and/or about 50% of a mixture. In other embodiments,any one of two or more compounds in a mixture may represent about: 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 65%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% of a mixture.

Although antioxidants cannot cancel the negative effects of PUFAperoxidation due to the stochastic nature of the process and thestability of PUFA peroxidation products (reactive carbonyls) toantioxidant treatment, co-administration of antioxidants withcompositions resistant to oxidation, such as those described herein, mayprove beneficial for treating oxidative stress-related disorders.

Certain antioxidants contemplated as useful for co-administrationinclude the following: vitamins, such as vitamin C and vitamin E;glutathione, lipoic acid, uric acid, carotenes, lycopene, lutein,anthocyanins, oxalic acid, phytic acid, tannins, coenzyme Q, melatonin,tocopherols, tocotrienols, polyphenols including resveratrol,flavonoids, selenium, eugenol, idebenone, mitoquinone, mitoquinol,ubiquinone, Szeto-Schiller peptides, and mitochondrial-targetedantioxidants. When not explicitly mentioned, quinone derivatives of theaforementioned antioxidants are also contemplated as useful forco-administration.

In some embodiments, stabilized compounds are administered withcompounds that upregulate antioxidant genes. In other embodiments,stabilized compounds are administered with compounds that affectsignaling pathways, such as the Keap1/Nrf2/ARE signaling pathway,thereby resulting in the production of anti-inflammatory and/orantioxidant proteins, such as heme oxygenase-1 (HO-1). In someembodiments, stabilized compounds are administered with antioxidantinflammation modulators. Antioxidant inflammation modulators suppresspro-oxidant and/or pro-inflammatory transcription factors. In someembodiments, antioxidant inflammation modulators are activators of thetranscription factor Nrf2. Nrf2 activation promotes the antioxidant,detoxification, and anti-inflammatory genes upregulation. In otherembodiments, antioxidant inflammation modulators suppress NF-κB. In someembodiments, antioxidant inflammation modulators suppress STAT3. Inother embodiments, stabilized compounds are administered with compoundsthat affect histone deacetylase activity. In some embodiments,stabilized compounds are administered with compounds that bind toantioxidant response elements (ARE). In other embodiments, stabilizedcompounds are administered with bardoxolone methyl(2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oic acid methyl ester) as theantioxidant inflammation modulator. In some embodiments, the antioxidantinflammation modulator is 2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oicacid, or a pharmaceutically acceptable ester thereof. In otherembodiments, the antioxidant inflammation modulator is an amide of2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oic acid. In some embodiments,the antioxidant inflammation modulator is a triterpenoid. In otherembodiments, the antioxidant inflammation modulator is selected from thefollowing compounds:

Additional antioxidants believed to be useful in co-administrationtherapies include those compounds disclosed in U.S. Pat. Nos. 6,331,532;7,179,928; 7,232,809; 7,888,334; 7,888,335; 7,432,305; 7,470,798; and7,514,461; and U.S. Patent Application Nos. 20020052342; 20030069208;20040106579; 20050043553; 20050245487; 20060229278; 20070238709;20070270381; 20080161267; 20080275005; 20090258841; 20100029706; and20110046219; in which the compounds disclosed therein are incorporatedby reference. These compounds are mitochondrially-targeted compounds andinclude, but are not limited to:

Compounds of Formulas I or II

wherein R₁ and R₂ are independently selected from —C₁-C₄ alkyl, —C₁-C₄haloalkyl, —CN, —F, —Cl, —Br, and —I; R₃ is selected from —C₁-C₄ alkyl,—C₁-C₄ haloakyl, —CN, —F, —Cl, and —I, and R₂₀ is independently selectedfrom —C₁-C₂₀ alkyl, —C₁-C₂₀ alkenyl, —C₁-C₂₀ alkynyl, and —C₁-C₂₀containing at least one double bond and at least one triple bond.

Compounds such as:3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester;3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chroman-2-yl)-propionicacid;2,2,-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-propano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester;2-Methyl-2-[3-(thiazol-2-ylsulfanyl)-propyl]-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;[3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonicacid dimethyl ester;[3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propyl]-phosphonicacid;3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-propionicacid methyl ester;4-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-2-yl)-butane-1-sulfonicacid dimethylamide;2-(3-Hydroxy-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;-(2-Chloro-ethyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;2-Methyl-2-thiazol-2-yl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-6-ol;2,2-Dimethyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;3-(6-Hydroxy-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-2-yl)-propionicacid;2-(3-Chloro-propyl)-2-methyl-3,4,7,8,9,10-hexahydro-7,10-ethano-2H-benzo[h]chromen-6-ol;4-(6-Hydroxy-2,2-dimethyl-3,4,7,8,9,10-hexahydro-7,10-methano-2H-benzo[h]chromen-5-ylmethylene)-2-methyl-5-propyl-2,4-dihydro-pyrazol-3-one.

Compounds such as: 2,2,7,8-Tetramethyl-5-phenyl-chroman-6-ol;4-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-yl)-benzoic acid methylester; 4-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-yl)-benzoic acid;2,2,7,8-Tetramethyl-5-pyridin-4-yl-chroman-6-ol;2,2,7,8-Tetramethyl-5-pyridin-3-yl-chroman-6-ol;5-(4-Methanesulfonyl-phenyl)-2,2,7,8-tetramethyl-chroman-6-ol;5-(4-Dimethylamino-phenyl)-2,2,7,8-tetramethyl-chroman-6-ol;5-(4-Chloro-phenyl)-2,2,7,8-tetramethyl-chroman-6-ol;4-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-yl)-benzene sulfonamide;5-(4-Methoxy-phenyl)-2,2,7,8-tetramethyl-chroman-6-ol;(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethyl)-1-hydroxyurea;2,2,7,8-Tetramethyl-5-(3-nitro-phenyl)-chroman-6-ol;2,2,7,8-Tetramethyl-5-(4-trifluoromethyl-phenyl)-chroman-6-ol;5-(4-tert-Butyl-phenyl)-2,2,7,8-tetramethyl-chroman-6-ol;2,2,7,8-Tetramethyl-5-(3,4,5-trimethoxy-phenyl)-chroman-6-ol;4-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-yl)-benzonitrile;5-(2,5-Dimethoxy-3,4-dimethyl-phenyl)-2,2,7,8-tetramethyl-chroman-6-ol;5-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-yl)-benzene-1,2,3-triol;5-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-yl)-2,3-dimethyl-benzene-1,4-diol;5-(2-Chloro-phenyl)-2,2,7,8-tetramethyl-chroman-6-ol;5-Furan-2-yl-2,2,7,8-tetramethyl-chroman-6-ol;5-Allylsulfanylmethyl-2,2,8-trimethyl-7-(3-methyl-butyl)-chroman-6-ol;5-Cyclopentylsulfanylmethyl-2,2,7,8-tetramethyl-chroman-6-ol;5-Hexylsulfanylmethyl-2,2,7,8-tetramethyl-chroman-6-ol;5-Allylsulfanylmethyl-2,2,7,8-tetramethyl-chroman-6-ol;5-(4,6-Dimethyl-pyrimidin-2-ysulfanylmethyl)-2,2,7,8-tetramethyl-chroman-6-ol;1-[3-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-yl-methylsulfanyl)-2-methyl-propionyl]-pyrrolidine-2-carboxylicacid;4-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-ylmethylene)-5-methyl-2-phenyl-2,4-dihydro-pyrazol-3-one;4-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-yl-methylene)-3-phenyl-4H-isoxazol-5-one;4-[4-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-yl-methylene)-3-methyl-5-oxo-4,5-dihydro-pyrazol-1-yl]-benzoicacid;4-(6-Hydroxy-2,2,7,8-tetramethyl-chroman-5-yl-methylene)-2-methyl-5-propyl-2,4-dihydro-pyrazol-3-one;5-Hydroxy-3-(6-hydroxy-2,2,7,8-tetramethyl-chroman-5-yl-methylene)-3H-benzofuran-2-one;2,5,7,8-Tetramethyl-2-thiophen-2-yl-chroman-6-ol;2-(2,5-Dimethyl-thiophen-3-yl)-2,5,7,8-tetramethyl-chroman-6-ol;2-(2,5-Dimethyl-thiophen-3-yl)-2,7,8-trimethyl-chroman-6-ol;8-Chloro-2-(2,5-dimethyl-thiophen-3-yl)-2,5,7-trimethyl-chroman-6-ol;5-Chloro-2,7,8-trimethyl-2-thiophen-2-yl-chroman-6-ol;5-[3-(6-Methoxymethoxy-2,7,8-trimethyl-chroman-2-yl)-propylidene]-thiazolidine-2,4-dione;5-[3-(6-Hydroxy-2,7,8-trimethyl-chroman-2-yl)-propylidene]-thiazolidine-2,4-dione;3-[6-Hydroxy-2,7,8-trimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-5-yl-methylsulfanyl]-2-methyl-propionicacid;2,7,8-Trimethyl-5-(5-methyl-1H-benzoimidazol-2-yl-sulfanylmethyl)-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol;2-[6-Hydroxy-2,7,8-trimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-5-ylmethylsulfanyl]-ethanesulfonicacid;5-(4,6-Dimethyl-pyrimidin-2-ylsulfanylmethyl)-2,7,8-trimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-6-ol;4-[2-(4,8-Dimethyl-tridecyl)-6-hydroxy-2,7,8-trimethyl-chroman-5-ylmethylsulfanyl]-benzoicacid;1-{3-[6-Hydroxy-2,7,8-trimethyl-2-(4,8,12-trimethyl-tridecyl)-chroman-5-ylmethylsulfanyl]-2-methyl-propionyl}-pyrrolidine-2-carboxylicacid; 2-(2,2-Dichloro-vinyl)-2,5,7,8-tetramethyl-chroman-6-ol;2-(2,2-Dibromo-vinyl)-2,5,7,8-tetramethyl-chroman-6-ol;5-(5-Chloro-3-methyl-pent-2-enyl)-2,2,7,8-tetramethyl-chroman-6-ol;5-Chloro-2-(2,5-dimethyl-thiophen-3-yl)-2,7,8-trimethyl-chroman-6-ol;2-(3-Chloro-propyl)-5,7-dimethyl-2-thiophen-2-yl-chroman-6-ol;5-Chloro-2-(2,5-dimethyl-thiazol-4-yl)-2,7,8-trimethyl-chroman-6-ol;5-Chloro-2-(2,5-dimethyl-thiazol-4-yl)-2,7,8-trimethyl-2H-chromen-6-ol;and 5-Chloro-2-(2,5-dimethyl-thiazol-4-yl)-2,7,8-trimethyl-chroman-6-ol.

Compounds such as: dimebolin(2,8-dimethyl-5-(2-(6-methylpyridin-3-yl)ethyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole),8-chloro-2-methyl-5-(2-(6-methylpyridin-3-yl)ethyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole,mebhydroline(5-benzyl-2-methyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole),2,8-dimethyl-1,3,4,4a,5,9b-hexahydro-1H-pyrido[4,3-b]indole,8-fluoro-2-(3-(pyridin-3-yl)propyl)-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole,and 8-methyl-1,3,4,4a,5,9b-tetrahydro-1H-pyrido[4,3-b]indole.

Compounds such as:2-(3-hydroxy-3-methylbutyl)-3,5-dimethyl-6-(4-(trifluoromethyl)phenyl)cyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-6-(4-methoxyphenyl)-3,5-dimethylcyclohexa-2,5-diene-1,4-dione;4-(5-(3-hydroxy-3-methylbutyl)-2,4-dimethyl-3,6-dioxocyclohexa-1,4-dienyl)benzonitrile;2-(3-hydroxy-3-methylbutyl)-3,5-dimethyl-6-(naphthalen-2-yl)cyclohexa-2,5-diene-1,4-dione;2-(3,4-difluorophenyl)-6-(3-hydroxy-3-methylbutyl)-3,5-dimethylcyclohexa-2,5-diene-1,4-dione;2-(4-fluorophenyl)-6-(3-hydroxy-3-methylbutyl)-3,5-dimethylcyclohexa-2,5-diene-1,4-dione;2-(4-chlorophenyl)-6-(3-hydroxy-3-methylbutyl)-3,5-dimethylcyclohexa-2,5-diene-1,4-dione;2-(2,3-dihydrobenzofuran-2-yl)-6-(3-hydroxy-3-methylbutyl)-3,5-dimethylcyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-phenethylcyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-phenylcyclohexa-2,5-diene-1,4-dione;2-benzyl-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(3-phenylpropyl)cyclohexa-2,5-diene-1,4-dione;2-(1-hydroxy-2-phenylethyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-3-(4-methoxyphenyl)-5,6-dimethyl-cyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(4-(trifluoromethyl)-phenyl)cyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(naphthalen-2-yl)cyclohexa-2,5-diene-1,4-dione;2-(benzofuran-2-yl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(4-chlorophenyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(4-ethylphenyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(3-(trifluoromethyl)phenyl)-cyclohexa-2,5-diene-1,4-dione;2-(4-tert-butylphenyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-cyclohexa-2,5-diene-1,4-dione;2-(4-fluorophenyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(3-fluorophenyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;4-(2-(3-hydroxy-3-methylbutyl)-4,5-dimethyl-3,6-dioxocyclohexa-1,4-dienyl)benzonitrile;2-(3,4-difluorophenyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-cyclohexa-2,5-diene-1,4-dione;2-(2-fluorophenyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-3-(3-methoxyphenyl)-5,6-dimethyl-cyclohexa-2,5-diene-1,4-dione;2-(4-fluoro-2-methoxyphenyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(benzo[d][1,3]dioxol-5-yl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(2,4-difluorophenyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-3-(4-methoxyphenyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(3,5-bis(trifluoromethyl)phenyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(4-chlorophenyl)-6-(3-hydroxy-3-methylbutyl)-3,5-dimethylcyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(2-(thiazol-2-yl)ethyl)cyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(2-(thiazol-5-yl)ethyl)cyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(2-(pyridin-2-yl)ethyl)cyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(2-(pyridazin-4-yl)ethyl)cyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(2-(thiophen-2-yl)ethyl)cyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(2-(thiophen-3-yl)ethyl)cyclohexa-2,5-diene-1,4-dione;2-(2-(furan-2-yl)ethyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(2-(furan-3-yl)ethyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(2-(1H-pyrazol-5-yl)ethyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(2-(1H-pyrazol-4-yl)ethyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(2-(1H-pyrazol-1-yl)ethyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(2-(1H-imidazol-5-yl)ethyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(2-(1H-imidazol-2-yl)ethyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(2-(oxazol-5-yl)ethyl)cyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(2-(oxazol-2-yl)ethyl)cyclohexa-2,5-diene-1,4-dione;2-(3-hydroxy-3-methylbutyl)-5,6-dimethyl-3-(2-(oxazol-4-yl)ethyl)cyclohexa-2,5-diene-1,4-dione;and2-(2-(1H-indol-3-yl)ethyl)-3-(3-hydroxy-3-methylbutyl)-5,6-dimethylcyclohexa-2,5-diene-1,4-dione.

Compounds such as:

wherein m is —C₁-C₂₀ alkyl, —C₁-C₂₀ alkenyl, —C₁-C₂₀ alkynyl, or —C₁-C₂₀containing at least one double bond and at least one triple bond, andthe counterion is a pharmaceutically acceptable anion.

Compounds such as:3-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)propyltriphenylphosphonium salts;4-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)butyltriphenylphosphonium salts;5-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)pentyltriphenylphosphonium salts;6-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)hexyltriphenylphosphonium salts;7-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)heptyltriphenylphosphonium salts;8-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)octyltriphenylphosphonium salts;9-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)nonyltriphenylphosphonium salts;10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyltriphenylphosphonium salts;11-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)undecyltriphenylphosphonium salts;12-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)dodecyltriphenylphosphonium salts;13-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)propyldecyltriphenylphosphonium salts;14-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)butyldecyltriphenylphosphonium salts;15-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)pentadecyltriphenylphosphonium salts;16-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)hexadecyltriphenylphosphonium salts;17-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)heptadecyltriphenylphosphonium salts;18-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)octadecyltriphenylphosphonium salts;19-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)nonadecyltriphenylphosphonium salts;20-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)icosyltriphenylphosphonium salts;3-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)propyltriphenylphosphonium salts;4-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)butyl triphenylphosphoniumsalts; 5-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)pentyltriphenylphosphonium salts;6-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)hexyl triphenylphosphoniumsalts; 7-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)heptyltriphenylphosphonium salts;8-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)octyl triphenylphosphoniumsalts; 9-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)nonyltriphenylphosphonium salts;10-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)decyltriphenylphosphonium salts;11-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)undecyltriphenylphosphonium salts;12-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)dodecyltriphenylphosphonium salts;13-(4,5-dimethoxy-2-methyl-3,6-dihydroxybenzyl)propyldecyltriphenylphosphonium salts;14-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)butyldecyltriphenylphosphonium salts;15-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)pentadecyltriphenylphosphonium salts;16-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)hexadecyltriphenylphosphonium salts;17-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)heptadecyltriphenylphosphonium salts;18-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)octadecyltriphenylphosphonium salts;19-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)nonadecyltriphenylphosphonium salts;20-(4,5-dimethoxy-2-methyl-3,6-dihydroxyphenyl)icosyltriphenylphosphonium salts; wherein the counterion of the salt is apharmaceutically acceptable anion such as bromide, methanesulfonateethanesulfonate, propanesulfonate, benzenesulfonate, p-toluenesulfonate,or 2-naphthylene sulfonate.

Additionally, it is contemplated that coadministration of antioxidantscould take the form of consuming foods known to have increased levels ofbeneficial antioxidants. Such foods include both regular foods and“superfoods” which contain antioxidants. These foods include fruits,vegetables, and other foodstuffs such as strawberries, blackcurrants,blackberries, oranges, blueberries, pomegranates, tea, coffee, oliveoil, chocolate, cinnamon, herbs, red wine, grain cereals, eggs, meat,legumes, nuts, spinach, turnip, rhubarb, cocao beans, maize, beans,cabbage, and the like.

Delivery and Additional Formulations:

It is well known that triglycerides are the main constituents ofvegetable oils and animal fats. It is also known that a triglyceride isan ester compound derived from glycerol and three fatty acids.Triglycerides are metabolized by enzymes such as lipases which hydrolyseester bonds and release fatty acids and glycerol. Indeed, thismetabolism releases fatty acids which can then be taken upon by cellsvia a fatty acid transporter protein. It is contemplated that PUFAs andPUFA mimetics that are useful in treating various diseases may beincorporated into fats such as triglycerides, diglycerides, and/ormonoglycerides for administration to a patient.

The delivery of the PUFAs, PUFA mimetics, PUFA pro-drugs, andtriglycerides containing PUFAs and/or PUFA mimetics could be through amodified diet. Alternatively, the PUFAs, PUFA mimetics, PUFA pro-drugs,and triglycerides containing PUFAs and/or PUFA mimetics can beadministered as foods or food supplements, on their own or as complexeswith ‘carriers’, including, but not limited to, complexes with albumin.

Other methods of delivering the reinforced PUFAs or their precursors,such as methods typically used for drug delivery and medicationdelivery, can also be employed. These methods include, but are notlimited to, peroral delivery, topical delivery, transmucosal deliverysuch as nasal delivery, nasal delivery through cribriform plate,intravenous delivery, subcutaneous delivery, inhalation, or through eyedrops.

Targeted delivery methods and sustained release methods, including, butnot limited to, the liposome delivery method, can also be employed.

It is contemplated that the isotopically modified compounds describedherein may be administered over a course of time, in which the cells andtissues of the subject will contain increasing levels of isotopicallymodified compounds over the course of time in which the compounds areadministered.

Compositions containing the active ingredient may be in a form suitablefor oral use, for example, as tablets, troches, lozenges, aqueous oroily suspensions, oil-in-water emulsions, dispersible powders orgranules, emulsions, hard or soft capsules, or syrups or elixirs. Suchcompositions may contain excipients such as bulking agents,solubilization agents, taste masking agents, stabilisers, colouringagents, preservatives and other agents known to those ordinarily skilledin the art of pharmaceutical formulation. In addition, oral forms mayinclude food or food supplements containing the compounds describedherein. In some embodiments supplements can be tailor-made so that onetype of PUFA, such as omega-3 or omega-6 fatty acids can be added tofood or used as a supplement depending on the dominant fat that the foodor the subject's diet contains. Moreover, compositions can betailor-made depending on the disease to be treated. For example, an LDLrelated condition may require more D-linoleic acid because cardiolipin,which is made of linoleic acid, is oxidized. In other embodiments, suchas retinal disease and neurological/CNS conditions may require moreomega-3 fatty acids such as D-linolenic acid, because D-omega-3 fattyacids are more relevant for treating these diseases. In some aspects,when the disease is associated with HNE, then D-omega-6 fatty acidsshould be prescribed, whereas for HHE, D-omega-3 fatty acids should beprescribed.

Compositions may also be suitable for delivery by topical application,as a spray, cream, ointment, lotion, or as a component or additive to apatch, bandage or wound dressing. In addition the compound can bedelivered to the site of the disease by mechanical means, or targeted tothe site of the disease through the use of systemic targetingtechnologies such as liposomes (with or without chemical modificationthat provides them with affinity for the diseased tissue), antibodies,aptamers, lectins, or chemical ligands such as albumin, with affinityfor aspects of the diseased tissue that are less abundant or not presenton normal tissue. In some aspects, topical application of cosmetics mayinclude the use of a carrier which is an isotopically modified compoundor mimetic described herein for delivering through skin such as by apatch. Eye disorders may be treated with eyedrops.

A pharmaceutical composition may also be in a form suitable foradministration by injection. Such compositions may be in the form of asolution, a suspension or an emulsion. Such compositions may includestabilizing agents, antimicrobial agents or other materials to improvethe function of the medicament. Some aspects of the invention alsoencompass dry or desiccated forms of the compounds which can readily beformed or reconstituted into a solution suspension or emulsion suitablefor administration by injection, or for oral or topical use. Delivery byinjection may be suitable for systemic delivery, and also local deliverysuch as injection into the eye for treating disorders relating to theeye.

Dosages

In some embodiments, compounds are dosed at about 0.01 mg/kg to about1000 mg/kg, about 0.1 mg/kg to about 100 mg/kg, and/or about 1 mg/kg toabout 10 mg/kg. In other embodiments, compounds are dosed at about:0.01, 0.1, 1.0, 5.0, 10, 25, 50, 75, 100, 150, 200, 300, 400, 500,and/or 1000 mg/kg.

EXAMPLES

Experimental: MALDI-TOF mass-spectra were recorded on a PE-ABI VoyagerElite delayed extraction instrument. Spectra were acquired with anaccelerating voltage of 25 KV and 100 ms delay in the positive ion mode.Unless otherwise specified, the ¹H NMR spectra were recorded on a VarianGemini 200 MHz spectrometer. HPLC was carried out on a Waters system.Chemicals were from Sigma-Aldrich Chemical Company (USA), Avocadoresearch chemicals (UK), Lancaster Synthesis Ltd (UK), and AcrosOrganics (Fisher Scientific, UK). Silica gel, TLC plates and solventswere from BDH/Merck. IR spectra were recorded with Vertex 70spectrometer. ¹H and ¹³C NMR spectra were obtained with a Bruker AC 400instrument at 400 and 100 MHz respectively, in CDCl₃ (TMS at δ=0.00 orCHCl₃ at δ=7.26 for ¹H and CHCl₃ at δ=77.0 for ¹³C as an internalstandard).

One of ordinary skill in the art will recognize that the below describedsyntheses can be readily modified to prepare additionaloxidation-resistant compounds. For example, one will recognize the esterof one type of stabilized compound can be cleaved to afford thecorresponding carboxylic acid. Likewise, carboxylic acids can be readilyconverted into additional derivatives, such as esters. Additionally, onewill appreciate that by varying the identity of the isotopically labeledstarting materials, isotopic variants of the below described compoundsmay be prepared. In the below described syntheses, paraformaldehyde-d₂is used as an isotopically labeled starting material. One will readilyappreciate that the same synthetic transformations can be used withparaformaldehyde-d₁, formaldehyde-d₁, paraformaldehyde-d₂,formaldehyde-d₂, and carbon-13 labeled variants of the aforementionedcompounds. Formaldehyde-d, is a well-characterized compound and isreadily available from known sources such as formic acid-d₁, formicacid-d₂, and/or dichloromethane-d, using generally known and understoodsynthetic transformations. Furthermore, radioactive analogues of thecompounds described herein can be prepared using tritium-containingstarting materials. These compounds would be useful for determiningincorporation in the cells and tissues of animals.

Example 1 Synthesis of 11,11-D2-linoleic acid

1,1-Dideutero-oct-2-yn-1-ol (2)

To a solution of ethylmagnesium bromide prepared from bromoethane (100ml), 1,2-dibromoethane (1 ml) and magnesium turnings (31.2 g) in dry THF(800 ml), heptyn-1 ((1); 170 ml) was added dropwise over 30-60 min underargon. The reaction mixture was stirred for 1 h, and thendeuteroparaform (30 g) was carefully added in one portion. The reactionmixture was gently refluxed for 2 h, chilled to −10° C., and then 5-7 mlof water was slowly added. The mixture was poured into 0.5 kg slurry ofcrushed ice and 40 ml concentrated sulphuric acid and washed with 0.5 Lof hexane. The organic phase was separated, and the remaining aqueousphase was extracted with 5:1 hexane:ethyl acetate (3×300 ml). Thecombined organic fraction was washed with sat. NaCl (1×50 ml), sat.NaHCO₃, (1×50 ml), and dried over Na₂SO₄. The solvent was evaporated invacuo to yield 119.3 g (99%) of colourless oil which was used withoutfurther purification. HRMS, m/z calculated for C₈H₁₂D₂O: 128.1168.found: 128.1173. ¹H NMR (CDCl₃, δ): 2.18 (t, J=7.0, 2H), 1.57 (s, 1H),1.47 (q, J=7.0 Hz, 2H), 1.31 (m, 4H), 0.87 (t, J=7.0 Hz, 3H).

1,1-Dideutero-1-bromo-oct-2-yne (3)

To a solution of (2) (3.48 g; 27.2 mmol) and pyridine (19 ml) in drydiethyl ether (300 ml), 36 ml of PBr₃ in 35 ml diethyl ether was addeddropwise with stirring over 30 min at −15° C. under argon. The reactionmixture was allowed to gradually warm up to r.t. and then refluxed 3 hwith stirring and 1 h without stirring. The reaction mixture was thencooled down to −10° C. and 500 ml of cold water was added. When theresidue dissolved, saturated NaCl (250 ml) and hexane (250 ml) wereadded, and the organic layer was separated. The aqueous fraction waswashed with hexane (2×100 ml), and the combined organic fractions werewashed with NaCl (2×100 ml) and dried over Na₂SO₄ in presence of tracesof hydroquinone and triethylamine. The solvent was removed bydistillation at atmospheric pressure followed by rotary evaporation. Theresidue was fractionated by vacuum distillation (3 mm Hg) to give 147.4g (82% counting per deutero-paraform) of pale yellow oil. B.p. 75° C.HRMS, m/z calculated for C₈H₁₁D₂Br: 190.0324. found: 189.0301, 191.0321.¹H NMR (CDCl₃, δ): 2.23 (t, J=7.0 Hz, 2H, CH₂), 1.50 (m, 2H, CH₂), 1.33(m, 4H, CH₂), 0.89 (t, J=6.9 Hz, 3H, CH₃),

11,11-Dideutero-octadeca-9,12-diynoic acid methyl ester (5)

CuI (133 g) was quickly added to 400 ml of DMF (freshly distilled overCaH₂), followed by dry NaI (106 g), K₂CO₃ (143 g). Dec-9-ynoic acidmethyl ester ((4); 65 g) was then added in one portion, followed bybromide (3) (67 g). Additional 250 ml of DMF was used to rinse thereagents off the flask walls into the bulk of reaction mixture, whichwas then stirred for 12 h. 500 ml of saturated aqueous NH₄Cl was thenadded with stirring, followed in a few minutes by saturated aqueous NaCland then by a 5:1 mixture of hexane:EtOAc (300 ml). The mixture wasfurther stirred for 15 min and then filtered through a fine mesh Schottglass filter. The residue was washed with hexane:EtOAc mix severaltimes. The organic fraction was separated, and the aqueous phase wasadditionally extracted (3×200 ml). The combined organic fraction wasdried (Na₂SO₄), traces of hydroquinone and diphenylamine were added, andthe solvent was evaporated in vacuo. The residue was immediatelydistilled at 1 mm Hg, to give 79 g (77%) of a 165-175° C. boilingfraction. HRMS, m/z calculated for C₁₉H₂₈D₂O₂: 292.2369. found:292.2365. ¹H NMR (CDCl₃, δ): 3.67 (s, 3H₂OCH₃), 2.3 (t, J=7.5 Hz, 2H,CH₂), 2.14 (t, J=7.0 Hz, 4H, CH₂), 1.63 (m, 2H, CH₂), 1.47 (m, 4H, CH₂),1.3 (m, 10H, CH₂), 0.88 (t, J=7.0 Hz, 3H, CH₃).

11,11-Dideutero-cis,cis-octadeca-9,12-dienoic acid methyl ester (6)

A suspension of nickel acetate tetrahydrate (31.5 g) in 96% EtOH (400ml) was heated with stirring to approx. 50-60° C. until the saltdissolved. The flask was flushed with hydrogen, and then 130 ml of NaBH₄solution, (prepared by a 15 min stirring of NaBH₄ suspension (7.2 g) inEtOH (170 ml) followed by filtering) was added dropwise over 20-30 minwith stirring. In 15-20 min ethylenediamine (39 ml) was added in oneportion, followed in 5 min by an addition of (5) (75 g) in EtOH (200ml). The reaction mixture was very vigorously stirred under hydrogen (1atm). The absorption of hydrogen stopped in about 2 h. To the reactionmixture, 900 ml of hexane and 55 ml of ice cold AcOH were added,followed by water (15 ml). Hexane (400 ml) was added, and the mixturewas allowed to separate. Aqueous fractions were extracted by 5:1 mix ofhexane:EtOAc. The completion of extraction was monitored by TLC. Thecombined organic phase was washed with diluted solution of H₂SO₄,followed by saturated NaHCO₃ and saturated NaCl, and then dried overNa₂SO₄. The solvent was removed at reduced pressure. Silica gel (Silicagel 60, Merck; 162 g) was added to a solution of silver nitrate (43 g)in anhydrous MeCN (360 ml), and the solvent removed on a rotavap. Theobtained impregnated silica gel was dried for 3 h at 50° C. (aspirationpump) and then 8 h on an oil pump. 30 g of this silica was used per gramof product. The reaction mixture was dissolved in a small volume ofhexane and applied to the silver-modified silica gel, and pre-washedwith a 1-3% gradient of EtOAc. When the non-polar contaminants werewashed off (control by TLC), the product was eluted with 10% EtOAc andthe solvent evaporated in vacuo to give 52 g of the title ester (6) as acolourless liquid. HRMS, m/z calculated for C₁₉H₃₂D₂O₂: 296.2682. found:296.2676. IR (CCl₄): {tilde over (ν)}=1740 cm⁻¹. ¹H NMR (CDCl₃, δ): 5.32(m, 4H), 3.66 (s, 3H, OCH₃), 2.29 (t, J=7.5 Hz, 2H, CH₂), 2.02 (m, 4H,CH₂), 1.60 (m, 2H, CH₂), 1.30 (m, 14H, CH₂), 0.88 (t, J=7.0 Hz, 3H,CH₃).

11,11-Dideutero-cis,cis-octadeca-9,12-dienoic acid (7)

A solution of KOH (46 g) in water (115 ml) was added to a solution ofester (6) (46 g) in MeOH (60 ml). The reaction mixture was stirred at40-50° C. for 2 h (control by TLC) and then diluted with 200 ml ofwater. Two thirds of the solvent were removed (rotavap). Dilutedsulphuric acid was added to the residue to pH 2, followed by diethylether with a little pentane. The organic layer was separated and theaqueous layer washed with diethyl ether with a little pentane. Thecombined organic fractions were washed with saturated aqueous NaCl andthen dried over Na₂SO₄. The solvent was evaporated to give 43 g of (7)(99%). IR (CCl₄): {tilde over (ν)}=1741, 1711 cm⁻.

Example 2 Synthesis of 11,11,14,14-D4-linolenic acid

1,1-Dideutero-pent-2-yn-1-ol (9)

But-1-yne (8) was slowly bubbled through a solution of ethylmagnesiumbromide prepared from bromoethane (100 ml) and magnesium turnings (31.3g) in dry THF (800 ml) on a bath (−5° C.). Every now and then thebubbling was stopped and the cylinder with but-1-yne was weighed tomeasure the rate of consumption. The supply of alkyne was stoppedshortly after a voluminous precipitate formed (the measured mass ofalkyne consumed was 125 g). The reaction mixture was warmed up to r.t.over 30 min, and then stirred for 15 min. The mixture was then heated upto 30° C., at which point the precipitate dissolved, and then stirred atr.t. for another 30 min. Deuteroparaform (28 g) was added in one portionand the mixture was refluxed for 3 h, forming a clear solution. It wascooled down to r.t. and poured into a mixture of crushed ice (800 g) and50 ml conc. H₂SO₄. Hexane (400 ml) was added and the organic layer wasseparated. The aqueous phase was saturated with NaCl and extracted witha 4:1 mixture of hexane:EtOAc (1 L). The completion of extractionprocess was monitored by TLC. The combined organic phases were washedwith saturated NaCl, NaHCO₃ and again NaCl, and dried over Na₂SO₄. Thesolvent was removed by distillation at the atmospheric pressure (maxvapour temperature 105° C.). The residue (70.5 g; 94%) was used withoutfurther purification. HRMS, m/z calculated for C₅H₆D₂O: δ6.0699. found:86.0751. ¹H NMR (CDCl₃, δ): 2.21 (q, J=7.5 Hz, 2H, CH₂), 1.93 (br s, 1H,OH), 1.12 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 87.7, 77.6, 13.7,12.3 (signal of CD₂ is absent).

1,1-Dideutero-1-bromo-pent-2-yne (10)

To a solution of (9) (70.5 g) and pyridine (16.5 ml) in dry diethylether (280 ml), 32.3 ml of PBr₃ in 50 ml diethyl ether was addeddropwise with stirring over 30 min at −10° C. under argon. The reactionmixture was allowed to gradually warm up to r.t. over 1 h. A smallamount of hydroquinone was added, and the mixture was then refluxed for4.5 h. The reaction mixture was then cooled down to −10° C. and 350 mlof cold water was added. When the residue dissolved, saturated NaCl (350ml) and hexane (300 ml) were added, and the organic layer was separated.The aqueous fraction was washed with diethyl ether (2×150 ml), and thecombined organic fractions were washed with NaCl (2×50 ml) and driedover Na₂SO₄ in presence of traces of hydroquinone and triethylamine. Thesolvent was removed at atmospheric pressure, and then the 147-155° C.boiling fraction was distilled off. Alternatively, upon reaching 100°C., the distillation at atmospheric pressure was stopped and the productdistilled off at 77-84° C. (25 mm Hg). Yield: 107 g of clear liquid.HRMS, m/z calculated for C₅H₅D₂Br: 147.9855. found: 146.9814, 148.9835.IR (CCl₄): {tilde over (ν)}=2251 cm⁻¹. ¹H NMR (CDCl₃, δ): 2.23 (q, J=7.5Hz, 2H, CH₂), 1.11 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 89.3,74.5, 13.4, 12.6 (signal of CD₂ is absent).

1,1,4,4-Tetradeutero-octa-2,5-diyn-1-ol (12)

Ethylmagnesium bromide, prepared from ethyl bromide (53 ml) andmagnesium turnings (15.8 g) in 400 ml of dry THF, was added in smallportions to 350 ml of dry THF, simultaneously with acetylene bubblingthrough this mixture (at approx. 25 L/h rate) with vigorous stirring.The Grignard reagent solution was fed to the mixture at approx. 10 mlper 2-5 min. When all ethylmagnesium bromide was added (after approx.2.5 h), acetylene was bubbled through the system for another 15 min.Deuteroparaform (17.3 g) and CuCl (0.2 g) were added under argon, andthe reaction mixture was refluxed without stirring for 2.5 h, untildeuteroparaform dissolved, to yield a solution of (11). Ethylmagnesiumbromide solution, prepared from 14.8 g magnesium and 50 ml ethyl bromidein 250 ml of dry THF, was added dropwise to the reaction mixture over 20min. When the gas emanation ceased, a condenser was attached and 250 mlof solvent were distilled off. The reaction mixture was then cooled to30° C., and CuCl (1.4 g) was added followed by a dropwise addition, over15 min, of bromide (10) (69 g). The reaction mixture was then refluxedfor 5 h, cooled slightly (a precipitate will form if cooling is toofast), and poured into a slurry of crushed ice (1-1.2 kg) and 40 mlconcentrated H₂SO₄. The mixture was washed with hexane (600 ml). Theorganic fraction was separated, and the aqueous fraction wasadditionally extracted with 5:1 hexane:EtOAc (2×400 ml). The combinedorganic fraction was washed, with saturated NaCl, followed by saturatedNaHCO₃ and NaCl. The bulk of the solvent was removed at atmosphericpressure in presence of traces of hydroquinone and triethylamine. Theresidue was flushed through 100 ml of silica gel (eluent: 7:1hexane:EtOAc). The bulk of the solvent was removed at the atmosphericpressure, and the remainder on a rotavap. 49.5 g (85%) of the titlecompound obtained was used without further purification. HRMS, m/zcalculated for C₈H₆D₄O: 126.0979. found: 126.0899. IR(CCl₄): {tilde over(ν)}=3622 cm⁻¹. ¹H NMR (CDCl₃, δ): 2.16 (q, J=7.5 Hz, 2H, CH₂), 1.85 (brs, 1H, OH), 1.11 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 82.3, 80.4,78.3, 72.6, 13.7, 12.2

1,1,4,4-Tetradeutero-1-bromo-octa-2,5-diyne (13)

was synthesized as described for bromide (3); 2 ml of pyridine, 14 mlPBr₃ and 250 ml of diethyl ether was used for 54.2 g of alcohol (12).The product was purified by distillation at 4 mm Hg. Yield: 53 g (65%)of (13); b.p. 100-110° C. HRMS, m/z calculated for C₈H₅D₄Br: 188.0135.found: 187.0136, 189.0143. IR (CCl₄): {tilde over (ν)}=2255 cm⁻¹. ¹H NMR(CDCl₃, δ): 2.13 (q, J=7.5 Hz, 2H, CH₂); 1.07 (t, J=7.5 Hz, 3H, CH₃).¹³C NMR (CDCl₃, δ): 82.5, 81.8, 75.0, 72.0, 13.6, 12.2.

11,11,14,14-Tetradeutero-octadeca-8,12,15-triynoic acid methyl ester(15)

was synthesized in a way similar to that described for11,11-dideutero-octadeca-9,12-diynoic acid methyl ester (5). CuI (97 g)was quickly added to 400 ml of DMF (freshly distilled over CaH₂),followed by dry NaI (77.5 g), K₂CO₃ (104.5 g). Dec-9-ynoic acid methylester ((14); 47.5 g) was then added in one portion, followed by bromide(13) (48.5 g). Additional 250 ml of DMF was used to rinse the reagentsoff the flask walls into the bulk of reaction mixture, which was thenstirred for 12 h. 500 ml of saturated aqueous NH₄Cl was then added withstirring, followed in a few minutes by saturated aqueous NaCl (300 ml)followed by a 5:1 mixture of hexane:EtOAc (300 ml). The mixture wasfurther stirred for 15 min and then filtered through a fine mesh Schottglass filter. The residue was washed with hexane:EtOAc mix severaltimes. The organic fraction was separated, and the aqueous phase wasadditionally extracted (3×200 ml). The combined organic fraction wasdried (Na₂SO₄), traces of hydroquinone and diphenylamine were added, andthe solvent was evaporated in vacuo. The residue was immediatelydistilled at 1 mm Hg, to give 45.8 g (62%) of a 173-180° C. boilingfraction. An additional crystallisation was carried out as follows. Theester (15) was dissolved in hexane (500 ml) and cooled down to −50° C.The crystals formed were washed in cold hexane. The yield of this stepis 80%. HRMS, m/z calculated for C₁₉H₂₂D₄O₂: 290.2180. found: 290.2200.¹H NMR (CDCl₃, δ): 3.66 (s, 3H, OCH₃), 2.29 (t, J=7.5 Hz, 2H, CH₂), 2.15(m, 4H, CH₂), 1.61 (m, 2H, CH₂), 1.47 (m, 2H, CH₂), 1.30 (m, 6H, CH₂),1.11 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 174.1, 82.0, 80.6,74.7, 74.6, 73.7, 73.0, 51.3, 33.9, 28.9, 28.6, 28.52, 28.49, 24.8,18.5, 13.7, 12.2.

11,11,14,14-Tetradeutero-cis,cis,cis-octadeca-8,12,15-trienoic acidmethyl ester (16)

was synthesized in a way similar to that described for11,11-Dideutero-cis,cis-octadeca-9,12-dienoic acid methyl ester (‘6’). Asuspension of nickel acetate tetrahydrate (42 g) in 96% EtOH (400 ml)was heated with stirring to approx. 50-60° C. until the salt dissolved.The flask was flushed with hydrogen, and then 130 ml of NaBH₄ solution,(prepared by a 15 min stirring of NaBH₄ suspension (7.2 g) in EtOH (170ml) followed by filtering) was added dropwise over 20-30 min withstirring. In 15-20 min ethylenediamine (52 ml) was added in one portion,followed in 5 min by an addition of (15) (73 g) in EtOH (200 ml). Thereaction mixture was very vigorously stirred under hydrogen (1 atm). Theabsorption of hydrogen stopped in about 2 h. To the reaction mixture,900 ml of hexane and 55 ml of ice cold AcOH were added, followed bywater (15 ml). Hexane (400 ml) was added, and the mixture was allowed toseparate. Aqueous fractions were extracted by 5:1 mix of hexane:EtOAc.The completion of extraction was monitored by TLC. The combined organicphase was washed with diluted solution of H₂SO₄, followed by saturatedNaHCO₃ and saturated NaCl, and then dried over Na₂SO₄. The solvent wasremoved at reduced pressure. Silica gel for purification was prepared asdescribed for (6). 30 g of this silica was used per gram of product. Thereaction mixture was dissolved in a small volume of hexane and appliedto the silver-modified silica gel, and pre-washed with a 1-5% gradientof EtOAc. When the non-polar contaminants were washed off (control byTLC), the product was eluted with 10% EtOAc and the solvent evaporatedin vacuo to give 42 g of the title ester (16) as a colourless liquid.HRMS, m/z calculated for C₁₉H₂₈D₄O₂: 296.2649. found: 296.2652. IR(CCl₄): {tilde over (ν)}=1740 cm⁻¹. ¹H NMR (CDCl₃, δ): 5.4 (m, 6H, CH—double bond), 3.68 (s, 3H, OCH₃), 2.33 (t, J=7.5 Hz, 2H, CH₂), 2.09 (m,4H, CH₂), 1.62 (m, 2H, CH₂), 1.33 (m, 8H, CH₂), 0.97 (t, J=7.5 Hz, 3H,CH₃). ¹³C NMR (CDCl₃, δ): 174.1, 131.9, 130.2, 128.2, 128.1, 127.7,126.9, 51.3, 34.0, 29.5, 29.04, 29.02, 27.1, 25.5, 24.9, 20.5, 14.2.

11,11,14,14-Tetradeutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (17)

A solution of KOH (1.5 g, 27 mmol) in water (2.6 ml was added to asolution of ester (16) (1.00 g, 3.4 mmol) in MeOH (15 ml). The reactionmixture was stirred at 40-50° C. for 2 h (control by TLC) and thendiluted with 20 ml of water. Two thirds of the solvent were removed(rotavap). Diluted sulfuric acid was added to the residue to pH 2,followed by diethyl ether with a little pentane (50 ml). The organiclayer was separated and the aqueous layer washed with diethyl ether witha little pentane (3×30 ml). The combined organic fractions were washedwith saturated aqueous NaCl and then dried over Na₂SO₄. The solvent wasevaporated to give 0.95 g of (17) (100%). IR (CCl₄): {tilde over(ν)}=1741, 1711 cm⁻¹.

Example 3 Synthesis of 14,14-D2-linolenic acid

4,4-Dideutero-octa-2,5-diyn-1-ol (19)

To a solution of ethylmagnesium bromide, prepared from ethyl bromide(9.2 ml, 123.4 mmol) and magnesium turnings (2.74 g, 112.8 mmol) in 40ml of dry THF, on an ice bath with stirring, propargyl alcohol (3.16 g,56.4 mmol) in THF (5 ml) was added dropwise over 10-15 min. The reactionmixture was allowed to warm up to r.t. and stirred for another 2 h, withoccasional warming to 40° C. To thus generated dianion, 0.13 g of CuClwas added, followed by slow (over 15 min) addition of bromide (10) (6.9g) in THF (20 ml). The reaction mixture was then stirred for 1 h at r.t.and then refluxed for 5 h. The reaction mixture was then refluxed for 5h, cooled slightly (a precipitate will form if cooling is too fast), andpoured into a slurry of crushed ice and 2.5 ml concentrated H2SO4. Themixture was washed with hexane (600 ml). The organic fraction wasseparated, and the aqueous fraction was additionally extracted with 5:1hexane:EtOAc. The combined organic fraction was washed, with saturatedNaCl, followed by saturated NaHCO3 and NaCl, and dried over Na2SO4. Thebulk of the solvent was removed at atmospheric pressure in presence oftraces of hydroquinone and triethylamine. The product was purified by CC(hexane:EtOAc=15:1) to give 3.45 g (59%) of the product 19. HRMS, m/zcalculated for C₈H₈D2O: 124.0855. found: 124.0849. IR (CCl₄): {tildeover (ν)}=3622 cm⁻¹. ¹H NMR (CDCl₃, δ): 4.21 (m, 2H, CH2), 2.4 (m, 1H,OH), 2.16 (q, J=7.5 Hz, 2H, CH2), 1.11 (t, J=7.5 Hz, 3H, CH3). ¹³C NMR(CDCl₃, δ): 82.3, 80.4, 78.3, 72.6, 51.0, 13.7, 12.2.

4,4-Dideutero-1-bromo-octa-2,5-diyne (20)

was synthesized as described for (3), except all solvent was removed ona rotavap. From 3.4 g (27 mmol) of (19), 3.9 g (75%) of the bromide (20)was obtained, which was used without further purification. HRMS, m/zcalculated for C₈H₇D₂Br: 186.0011. found: 185.0019, 187.0012. IR (CCl₄):{tilde over (ν)}=2255 cm⁻¹. ¹H NMR (CDCl₃, δ): 3.88 (br s, 2H, CH₂),2.13 (q, J=7.5 Hz, 2H, CH₂), 1.07 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR(CDCl₃, δ): 82.5, 81.8, 75.0, 72.0, 14.8, 13.6, 12.2.

14,14-Dideutero-octadeca-8,12,15-triynoic acid methyl ester (21)

was synthesized as described for (5). The product obtained from 9.7 gCuI, 7.8 g NaI, 10.5 g K₂CO₃, 4.85 g of bromide (20), 4.75 g of methylester (14) and 40 ml of anhydrous DMF, was purified by CC (25:1hexane:EtOAc) to give 4.5 g (60%) of the title compound. HRMS, m/zcalculated for C₁₉H₂₄D₂O₂: 288.2056. found: 288.2046. ¹H NMR (CDCl₃, δ):3.66 (s, 3H, OCH₃), 3.12 (m, 2H, CH₂), 2.29 (t, J=7.5 Hz, 2H, CH₂), 2.15(m, 4H, CH₂), 1.61 (m, 2H, CH₂), 1.47 (m, 2H, CH₂), 1.30 (m, 6H, CH₂),1.11 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 174.1, 82.0, 80.6,74.7, 74.6, 73.7, 73.0, 51.3, 33.9, 28.9, 28.6, 28.52, 28.49, 24.8,18.5, 13.7, 12.2, 9.7.

14,14-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid methyl ester(22)

was synthesized as described for the linoleic acid derivative (6). For areduction of 4.5 g of (21), 2.6 g of nickel acetate tetrahydrate and 3.2ml ethylenediamine was used. The product was purified onAgNO₃-impregnated silica gel as described for (6). HRMS, m/z calculatedfor C₁₉H₃₀D₂O₂: 294.2526. found: 294.2529. IR (CCl₄): {tilde over(ν)}=1740 cm⁻¹. ¹H NMR (CDCl₃, δ): 5.37 (m, 6H, CH-double bond), 3.68(s, 3H, OCH₃), 2.82 (m, 2H, CH₂), 2.33 (t, J=7.5 Hz, 2H, CH₂), 2.09 (m,4H, CH₂), 1.62 (m, 2H, CH₂), 1.33 (m, 8H, CH₂), 0.97 (t, J=7.5 Hz, 3H,CH₃). ¹³C NMR (CDCl₃, δ): 174.1, 131.9, 130.2, 128.2, 128.1, 127.7,126.9, 51.3, 34.0, 29.5, 29.1, 29.04, 29.02, 27.1, 25.5, 24.9, 20.5,14.2.

14,14-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (23)

To a solution of (22) (1 g, 3.4 mmol) in MeOH (15 ml), a solution of KOH(1.5 g, 27 mmol) in water (2.6 ml) was added in one portion. Thereaction mixture was then processed as described for (7) to yield 0.94 g(99%) of the title acid. IR (CCl₄): {tilde over (ν)}=1741, 1711 cm⁻¹.

Example 4 Synthesis of 11,11-D2-linolenic acid

Pent-2-yn-1-ol (24)

Butyn-1 ((8); 10.4 g) was bubbled through an ice-cold solution preparedfrom bromoethane (11.2 ml) and magnesium turnings (3.6 g) in THF (100ml). The reaction mixture was allowed to warm up to r.t. and thenstirred for 15 min. The mixture was then heated up to 30° C., at whichpoint all precipitate dissolved. The heating was removed and the mixturestirred for another 30 min, and then paraform (3 g) was added in oneportion. The reaction mixture was refluxed for 3 h (all paraformdissolved), then cooled to r.t., poured into a mixture of crushed ice(80 g) and 8 ml conc. H₂SO₄, and extracted with diethyl ether. Theorganic phase was washed with saturated NaHCO₃ and NaCl, and dried overNa₂SO₄. The solvent was removed on a rotavap, and the residue (7.56 g;90%) was used without further purification. HRMS, m/z calculated forC₅H₈O: 84.0575. found: 84.0583.

1-Bromo-pent-2-yne (25)

To a solution of (24) (11.7 g) and pyridine (2.66 ml) in dry diethylether (34 ml), 5.2 ml of PBr₃ in 5 ml diethyl ether was added dropwisewith stirring over 30 min at −10° C. under argon. The reaction mixturewas allowed to gradually warm up to r.t. over 1 h. A catalytic amount ofhydroquinone was added, and the mixture was then refluxed for 4.5 h. Thereaction mixture was then cooled down to −10° C. and 35 ml of cold waterwas added. When the residue dissolved, saturated NaCl (35 ml) anddiethyl ether (30 ml) were added, and the organic layer was separated.The aqueous fraction was washed with diethyl ether (2×15 ml), and thecombined organic fractions were washed with NaCl (2×400 ml) and driedover MgSO₄. The solvent was removed at atmospheric pressure, and thenunder reduced pressure (25 mm Hg), the 60-90° C. fraction was collected.Yield: 11.1 g (84%). HRMS, m/z calculated for C₅H₇Br: 145.9731. found:144.9750, 146.9757.

1,1-Dideutero-octa-2,5-diyn-1-ol (26)

was synthesized as described for (12) with 87% yield. HRMS, m/zcalculated for C₈H₈D₂O: 124.0855. found: 124.0868. IR (CCl₄): {tildeover (ν)}=3622 cm⁻¹. ¹H NMR (CDCl₃, δ): 2.65 (m, 2H, CH₂), 2.4 (m, 1H,OH), 2.1 (q, 2H, CH₂), 1.09 (t, 3H, CH₃).

1,1-Dideutero-1-bromo-octa-2,5-diyne (27)

was synthesized as described for (3), except all solvent was removed ona rotavap. The product was purified by distillation at reduced pressure.Yield: 86% (b.p. 100-105° C. at 4 mm Hg). HRMS, m/z calculated forC₈H₇D₂Br: 186.0011. found: 184.9948, 187.9999. IR (CCl₄): {tilde over(ν)}=2255 cm⁻¹. ¹H NMR (CDCl₃, δ): 2.66 (m, 2H, CH₂), 2.1 (q, 2H, CH₂),1.09 (t, 3H, CH₃).

11,11-Dideutero-octadeca-8,12,15-triynoic acid methyl ester (28)

was synthesized as described for (5). The product obtained from 7.1 gCuI, 5.66 g NaI, 7.65 g K₂CO₃, 3.55 g of bromide (27), 3.47 g of methylester (14) and 30 ml of anhydrous DMF, was purified by CC (25:1hexane:EtOAc) to give 3.7 g of the title compound. HRMS, m/z calculatedfor C₁₉H₂₄D₂O₂: 288.2056. found: 288.2069. ¹H NMR (CDCl₃, δ): 3.7 (s,3H, OCH₃), 3.15 (br. s, 2H, CH₂), 2.35 (m, 2H, CH₂), 2.17 (m, 4H, CH₂),1.61 (m, 2H, CH₂), 1.48 (m, 2H, CH₂), 1.35 (m, 6H, CH₂), 1.11 (t, 3H,CH₃).

11,11-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid methyl ester(29)

was synthesized as described for the linoleic acid derivative (6). For areduction of 3.7 g of (28), 2.16 g of nickel acetate tetrahydrate and2.62 ml ethylenediamine was used. The product was purified onAgNO₃-impregnated silica gel as described for (6) to give 1.5 g. HRMS,m/z calculated for C₁₉H₃₀D₂O₂: 294.2526. found: 294.2402. IR (CCl₄):{tilde over (ν)}=1740 cm⁻¹. ¹H NMR (CDCl₃, δ): 5.37 (m, 6H, CH-doublebond), 3.6 (s, 3H, OCH₃), 2.82 (m, 2H, CH₂), 2.33 (t, o=7.5 Hz, 2H,CH₂), 2.09 (m 4H, CH₂), 1.62 (m, 2H, CH₂), 1.33 (m, 8H, CH₂), 0.97 (t,J=7.5 Hz, 3H, CH₃). ¹³C NMR (CDCl₃, δ): 174.1, 131.9, 130.2, 128.2,128.1, 127.7, 126.9, 51.3, 34.0, 29.5, 29.1, 29.04, 29.02, 27.1, 25.5,24.9, 20.5, 14.2.

11,11-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (30)

To a solution of (29) (1.5 g, 5.1 mmol) in MeOH (7.5 ml), a solution ofKOH (1.5 g, 27 mmol) in water (3 ml) was added in one portion. Thereaction mixture was then processed as described for (17) to yield 0.9 gof the title acid. IR (CCl₄): {tilde over (ν)}=1741, 1711 cm⁻¹. ¹H NMR(CDCl₃, δ): 11.2 (br s, 1H, COOH), 5.37 (m, 6H, CH-double bond), 2.83(m, 2H, CH₂), 2.35 (t, J=7.5 Hz, 2H, CH₂), 2.06 (m 4H, CH₂), 1.63 (m,2H, CH₂), 1.32 (m, 8H, CH₂), 0.97 (t, J=7.5 Hz, 3H, CH₃). ¹³C NMR(CDCl₃, δ): 180.4, 131.9, 130.2, 128.3, 128.1, 127.6, 127.1, 34.1, 29.5,29.1, 29.03, 28.98, 27.2, 25.5, 24.6, 20.5, 14.2.

Example 5 Synthesis of 8,8-D₂-Linoleic Acid Methyl Ester

8-Hydroxyoctanoic acid (502).

A solution of 8-bromocaprylic acid (501, 37.5 g, 168 mmol), anhydroussodium acetate (60.0 g, 732 mmol) and sodium iodide (1.0 g, 6.7 mmol) inDMF (200 ml) was stirred at 110-120° C. for 8 h. The reaction mixturewas cooled to r.t., a solution of potassium hydroxide (28 g, 0.5 mol) inwater (150 ml), was added, and the mixture was stirred at 100° C. foranother hour. The reaction mixture was cooled to r.t. and poured intoslurry of ice and concentrated sulfuric acid (45 ml). The solutionobtained was saturated with NaCl and extracted (9×150 ml) with a mixtureof EtOAc and petroleum ether (1:1). Combined organic fractions werewashed twice with saturated NaCl and dried over Na₂SO₄. The solvent wasevaporated to give 26.5 g (98%) of the product which was used withoutfurther purification. A small amount of the product was further purifiedby CC on silica (eluent:petroleum ether:EtOAc=2:1) and characterized. ¹HNMR (400 MHz, CDCl₃) δ 1.27-1.39 (m, 6H), 1.50-1.68 (m, 4H), 2.32 (t,2H, J=7.5 Hz), 3.62 (t, 2H, J=6.5 Hz), 6.87 (br. s., 2H).

Methyl 8-(tetrahydro-2H-pyran-2-yloxy)octanoate (503).

8-Hydroxyoctanoic acid (502; 26.3 g, 164 mmol) was dissolved in methanol(500 ml) containing acetyl chloride (3.5 ml). The reaction mixture wasrefluxed for 5 h and the solvent removed in vacuo. To the residuedissolved in CH₂Cl₂ (200 ml), 3,4-dihydro-2H-pyran (29 ml, 318 mmol) wasadded, and the reaction mixture was refluxed for 20 min. Upon additionof 5 ml of triethylamine, the solvent was removed in vacuo, and theresidue was dissolved in petroleum ether (100 ml) and washed with water.The organic layer was flush-purified on a small silica column (silica,100 ml; eluent: from petroleum ether to petroleum ether:EtOAc=20:1). Thework-up yielded 38.2 g (90%) of the product which was used withoutfurther purification. A small amount of the product was further purifiedby CC on silica (eluent:petroleum ether:EtOAc=15:1) and characterized.IR (CCl₄): {tilde over (ν)}=1741 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ1.20-1.36 (m, 6H), 1.40-1.82 (m, 10H), 2.23 (t, 2H, J=7.5 Hz), 3.30 (dt,1H, J=9.5 Hz, 6.5 Hz), 3.39-3.46 (m, 1H), 3.59 (s, 3H), 3.65 (dt, 1H,J=9.5 Hz, 7.0 Hz), 3.76-3.83 (m, 1H), 4.47-4.52 (m, 1H).

[1,1-D₂]-8-(tetrahydro-2H-pyran-2-yloxy)octan-1-ol (504).

To a stirred solution of ester (503) (37.5 g, 145 mmol) in diethyl ether(100 ml) in an ice bath, a suspension of LiAlD₄ (4.0 g, 95 mmol) indiethyl ether (300 ml) was added drop wise over 1 h. To the coldreaction mixture, water (4 ml), 15% NaOH (4 ml) and water (12 ml) wereadded with stirring. The precipitate was filtered and washed with ethylether. Evaporation in vacuo gave 33.5 g (99%) of the product. A smallamount of the product was further purified by CC on silica(eluent:petroleum ether:EtOAc=10:1) and characterized. IR (CCl₄): {tildeover (ν)}=3638, 3499 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ 1.22-1.33 (m, 8H),1.42-1.56 (m, 8H), 1.61-1.69 (m, 1H), 1.71-1.80 (m, 1H), 2.38 (br. s.,1H), 3.31 (dt, 1H, J=9.5 Hz, 6.5 Hz), 3.40-3.46 (m, 1H), 3.66 (dt, 1H,J=9.5 Hz, 7.0 Hz), 3.76-3.84 (m, 1H), 4.49-4.53 (m, 1H). ¹³C NMR (100MHz, CDCl₃) δ 19.5, 25.3, 25.5, 26.0, 29.2, 29.3, 29.5, 30.6, 32.4,62.1, 67.5, 98.7.

[1,1-D₂]-8-(tetrahydro-2H-pyran-2-yloxy)octyl methanesulfonate (505).

To a solution of alcohol (504) (33.4 g, 144 mmol) and triethylamine (45ml, 323 mmol) in diethyl ether (300 ml) at 0° C., a solution of MsCl(14.2 ml, 183 mmol) in diethyl ether (100 ml) was added drop wise over 1h with stirring. The reaction mixture was warmed up to r.t. and treatedwith water. The organic phase, combined with washings (2×50 ml) of theaqueous phase with Et₂O, was washed twice with saturated NaCl, driedover Na₂SO₄, and decanted. This was flush-purified on a small silicacolumn (silica, 100 ml; petroleum ether:EtOAc=10:1). The work-up yielded43.7 g (98%) of methanesulfonate (505). IR (CCl₄): {tilde over (ν)}=1739cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ 1.26-1.41 (m, 8H), 1.44-1.59 (m, 6H),1.63-1.84 (m, 4H), 2.97 (s, 3H), 3.32 (dt, 1H, J=9.5 Hz, 6.5 Hz),3.42-3.50 (m, 1H), 3.69 (dt, 1H, J=9.5 Hz, 7.0 Hz) 3.78-3.86 (m, 1H),4.52-4.56 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 19.6, 25.2, 25.4, 26.0,28.7, 28.8, 29.1, 29.5, 30.7, 37.2, 62.3, 67.4, 98.8.

2-([8,8-D₂]-dec-9-yne-1-yloxy)tetrahydro-2H-pyran (506).

Methanesulfonate (505) (43.5 g, 140 mmol) in DMSO (100 ml) was addeddropwise with stirring over 1 h to a suspension of aethylenediamine-lithium acetylenide complex (70 g, 0.76 mol) in DMSO(200 ml), and then the mixture was stirred for 90 min. Reaction mixturewas poured on ice, extracted (Et₂O, 3×150 ml), dried over Na₂SO₄ andevaporated. This was flush-purified on a small silica column (silica,100 ml; petroleum ether). Removal of solvent (rotavap) gave 25.3 g (75%)of the product. A small amount of the product was further purified by CCon silica (eluent:petroleum ether: EtOAc=25:1) and characterized. IR(CCl₄): {tilde over (ν)}=3314 cm⁻¹. ¹H NMR (400 MHz, CDCl₃) δ 1.21-1.38(m, 8H), 1.42-1.57 (m, 8H), 1.62-1.70 (m, 1H), 1.73-1.83 (m, 1H), 1.89(s, 1H), 3.32 (d.t., 1H, J=9.5 Hz, 6.5 Hz), 3.42-3.50 (m, 1H), 3.68(d.t., 1H, J=9.5 Hz, 7.0 Hz) 3.78-3.86 (m, 1H), 4.51-4.54 (m, 1H). ¹³CNMR (100 MHz, CDCl₃) δ 19.6, 25.4, 26.1, 28.1, 28.5, 28.9, 29.2, 29.6,30.6, 30.7, 62.1, 67.5, 68.0, 98.7.

[8,8-D₂]-dec-9-yne-1-ol (507).

Ether (506) (25 g, 104 mmol) was dissolved in methanol (300 ml)containing pyridinium para-toluenesulfonate (0.2 g). Reaction mixturewas refluxed for 3 h, quenched with Et₃N (1 ml), the solvent removed invacuo, the residue dissolved in petroleum ether and filtered through asmall amount of silica gel. The solvent was evaporated to give 15.4 g(95%) of the product. A small amount of the product was further purifiedby CC on silica (eluent:petroleum ether:EtOAc=15:1) and characterized.IR(CCl₄): {tilde over (ν)}=3638, 3508, 3314 cm⁻¹. ¹H NMR (400 MHz,CDCl₃) δ 1.22-1.40 (m, 8H), 1.42-1.56 (m, 4H), 1.91 (s, 1H), 2.29 (br.s., 1H), 3.59 (t, J=6.5 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 25.6, 28.1,28.5, 29.0, 29.2, 32.6, 62.8, 68.1, 84.6.

[8,8-D₂]-methyl dec-9-ynoate (508).

To a solution of chromium trioxide (24 g, 0.24 mol) and concentratedsulfuric acid (21 ml) in water (100 ml) in a two-neck round bottom flaskon water bath at 30° C. with stirring, a solution of alcohol (507) (15.5g, 99 mmol) in acetone (150 ml) was added dropwise over 90 min. Uponaddition, the reaction mixture was stirred for another 15 min, and theexcess of oxidizer was quenched with isopropyl alcohol. The mixture waspoured into cold water and extracted with diethyl ether (5×50 ml).Combined organic fractions were washed with saturated NaCl, dried overNa₂SO₄, filtered, and the solvent removed in vacuo. The residue wasdissolved in methanol (200 ml) and upon addition of concentratedsulfuric acid (1 ml) refluxed for 90 min. The acid was quenched withtriethylamine (6.5 ml, 47 mmol), the solvent removed in vacuo, and theresidue purified by CC on silica (eluent:petroleum ether:EtOAc=50:1) togive 12.6 g (69% counting per alcohol (507)) of ester (508). andcharacterized. IR (CCl₄): {tilde over (ν)}=3314, 1740 cm⁻¹. ¹H NMR (400MHz, CDCl₃) δ 1.19-1.38 (m, 6H), 1.41-1.48 (m, 2H), 1.51-1.61 (m, 2H),1.88 (s, 1H), 2.25 (t, J=7.5 Hz, 2H), 3.60 (s, 3H). ¹³C NMR (100 MHz,CDCl₃) δ 24.7, 28.0, 28.3, 28.6, 28.8, 33.9, 51.3, 68.1, 84.4, 174.0.

[8,8-D₂]-methyl octadeca-9,12-diynoate (510).

To DMF (20 ml) were added with stirring CuI (3.9 g, 20 mmol), followedby NaI (3.1 g, 21 mmol), K₂CO₃ (4.2 g, 30 mmol), ester (508) (1.9 g,10.3 mmol), and bromide (509) (2.04 g, 10.8 mmol, synthesized asdescribed in [2]). The reaction mixture was stirred at r.t. for 12 h.Saturated aqueous ammonium chloride (20 ml) was added to the mixture,followed by saturated NaCl (15 ml). The precipitate and the aqueousphase were washed with petroleum ether. The combined organic fractionswere washed with saturated sodium chloride, dried over Na₂SO₄ andevaporated in vacuo. The residue was purified by CC on silica(eluent:petroleum ether:EtOAc=50:1) to give 2.47 g (82%) of the product.¹H NMR (400 MHz, CDCl₃) δ 0.86 (t, J=7.0 Hz, 3H), 1.22-1.36 (m, 10H),1.40-1.50 (m, 4H), 1.55-1.64 (m, 2H), 2.09-2.15 (m, 2H), 2.28 (t, J=7.5Hz, 2H), 3.09 (t, J=2.5 Hz, 2H), 3.64 (s, 3H). ¹³C NMR (100 MHz, CDCl₃)δ 9.6, 13.9, 18.6, 22.1, 24.8, 28.3, 28.4, 28.5, 28.7, 28.9, 31.0, 34.0,51.4, 74.4, 74.5, 80.2, 80.4, 174.2.

[8,8-D₂]-octadeca-9,12-dienoate (511).

A suspension of finely ground Ni(Ac)₂×4H₂O (0.8 g, 3.2 mmol) in 96%ethanol (25 ml) was heated with stirring to 50-60° C. until the salt wasfully dissolved. The system was flushed with hydrogen, and then asolution of NaBH₄ (3.4 ml; obtained by 15 min stirring of NaBH₄suspension (0.53 g, 14 mmol) in ethanol (12 ml) followed by filteringthrough a fine filter) was added over 10 min. Evolvement of hydrogen wasobserved. In 15-20 min, ethylenediamine (1.65 ml, 25 mmol) was added tothe reaction mixture in one portion with stirring, followed by thesolution of (510) (2.4 g, 8.2 mmol) in ethanol (10 ml). The reactionmixture was vigorously stirred under hydrogen until there was no furtherabsorption of hydrogen, and then treated with acetic acid (2.3 ml),water (10 ml), and extracted with petroleum ether:EtOAc (5:1). Combinedorganic fractions were washed with 10% sulfuric acid (10 ml), then withsaturated sodium chloride, dried over Na₂SO₄, and the solvent wasremoved in vacuo. The residue was purified by CC on silica(eluent:petroleum ether:EtOAc=50:1) to give 2.33 g (96%) of the product.The product was then purified again by CC on silica impregnated with 20%AgNO₃ (eluent: petroleum ether to petroleum ether:EtOAc=2:1). 1.75 g(72%) of the product was obtained (97% purity by GC). ¹H NMR (400 MHz,CDCl₃) δ 0.88 (t, J=7.0 Hz, 3H), 1.20-1.40 (m, 14H), 1.55-1.66 (m, 2H),1.97-2.09 (m, 2H), 2.29 (t, J=7.5 Hz, 2H), 2.72-2.79 (m, 2H), 3.66 (s,3H), 5.28-5.41 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 14.0, 22.5, 24.9,25.6, 27.2, 29.00, 29.08, 29.13, 29.3, 29.4, 31.5, 34.1, 51.4, 127.9,128.0, 129.9, 130.2, 174.2.

Example 6 Synthesis of 11-D-Linoleic Acid

oct-2-yn-1-ol (13).

To a solution of oct-2-ynal [See Corey, E. J.; Schmidt, G. TetrahedronLett. 1979, 20, 399; Meyer, M. P.; Klinman, J. P. Tetrahedron Lett.2008, 49, 3600] ((612); 1.00 g, 8.1 mmol)) in ethanol (15 ml) cooled to0° C., 0.11 g (2.6 mmol) of NaBD₄ was added in portions over 5 min. Uponaddition, the solution was stirred for another 30 min, diluted withwater (100 ml), and then extracted with Et₂O (4×20 ml). The combinedorganic fractions were washed with saturated NaCl, dried (Na₂SO₄), andthe solvent was removed at reduced pressure. Alcohol 613 (0.85 g, 83%)was purified by column chromatography (silica gel, petroleum ether:EtOAc(15:1)). ¹H NMR (400 MHz, CDCl₃) δ 0.88 (t, J=7.0 Hz, 3H, CH₃), 1.32 (m,4H, CH₂), 1.49 (quint, J=7.0 Hz, 2H, CH₂), 1.81 (br s, 1H, OH), 2.19(td, J=7.0 Hz, 2.0 Hz, 2H, CH₂), 4.22 (m, 1H, CHD).

1-bromooct-2-yne (614)

was synthesized as described in [See Hill, Sh.; Hirano, K.; Shmanai, V.V.; Marbois, B. N.; Vidovic, D.; Bekish, A. V.; Kay, B.; Tse, V.; Fine,J.; Clarke, C. F.; Shchepinov, M. S. Free Radic. Biol. Med., 2011, 50(1), 130-138.]. ¹H NMR (400 MHz, CDCl₃) δ 0.89 (t, J=7.0 Hz, 3H, CH₃),1.32 (m, 4H, CH₂), 1.50 (quint, J=7.0 Hz, 2H, CH₂), 2.22 (td, J=7.0 Hz,2.0 Hz, 2H, CH₂), 3.91 (m, 1H, CHD).

[11-²H]-ethyloctadeca-9,12-diynoate (615).

was synthesized as described [See Meyer, M. P.; Klinman, J. P.Tetrahedron Lett. 2008, 49, 3600; Hill, Sh.; Hirano, K.; Shmanai, V. V.;Marbois, B. N.; Vidovic, D.; Bekish, A. V.; Kay, B.; Tse, V.; Fine, J.;Clarke, C. F.; Shchepinov, M. S. Free Radic. Biol. Med., 2011, 50 (1),130-138]. CuI (2 g, 10.5 mmol), NaI (1.58 g, 10.5 mmol), K₂CO₃ (2.1 g,15 mmol), ethyl dec-9-ynoate (1.02 g, 5.2 mmol) and bromide 614 (1.03 g,5.4 mmol) were added to DMF (10 ml) with stirring. The reaction mixturewas stirred at RT for 12 h, then NH₄Cl (10 ml) and NaCl (8 ml) wereadded and the stirring continued for another 5 min. The precipitate wasseparated and washed with petroleum ether. Organic layers wereseparated, and the aqueous layer was extracted with petroleum ether. Thecombined organic fractions were washed with saturated NaCl, dried(Na₂SO₄), and the solvent was removed at reduced pressure. Columnchromatography (silica gel, petroleum ether:EtOAc (15:1)) yielded 1.29 g(81%) of the product. ¹H NMR (400 MHz, CDCl₃) δ 0.89 (t, J=7.0 Hz, 3H,CH₃), 1.25 (t, J=7.0 Hz, 3H, CH₃CH₂O), 1.31 (m, 10H, CH₂), 1.49 (m, 4H,CH₂), 1.61 (m, 2H, CH₂), 2.15 (td, J=7.0 Hz, 2.0 Hz, 2H, CH₂ inpropargylic position), 2.28 (t, J=7.5 Hz, 2H, CH₂COOEt), 3.10 (m, 1H,CHD), 4.12 (q, J=7.0 Hz, 2H, OCH₂CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 9.6(t, J=19.0 Hz), 13.9, 14.1, 18.56, 18.57, 22.1, 24.8, 28.4, 28.6, 28.7,28.9, 28.9, 31.0, 34.2, 60.0, 74.3, 74.5, 80.2, 80.3, 173.7.

[11-²H]-linoleic acid (616)

A suspension of triturated nickel acetate tetrahydrate (0.4 g, 1.6 mmol)in 96% ethanol (12 ml) was heated at 50-60° C. with stirring until thesalt dissolved. The system was flushed with hydrogen, and then 1.7 ml ofNaBH₄ (obtained by 15-min stirring of a NaBH₄ suspension (0.27 g, 14mmol) in ethanol (6 ml) followed by sfiltering) was added over 10 min,with some gas bubbles evolving. In 15-20 min, ethylenediamine (0.8 ml,12 mmol) was added in one portion with stirring, followed in 5 min by asolution of diyne 615 (1.2 g, 3.9 mmol) in ethanol (5 ml). The reactionmixture was stirred vigorously until there was no more absorption ofhydrogen, and then treated with acetic acid (1.2 ml), water (10 ml) andextracted with a mixture of petroleum ether and EtOAc (5:1). Thecombined organic fractions were washed with 10% sulphuric acid (5 ml)and then with saturated NaCl, dried (Na₂SO₄), and the solvent wasremoved at reduced pressure. Column chromatography (silica gel,petroleum ether:EtOAc (50:1)) yielded 1.14 g (94%) of the product. Theproduct was additionally purified [3] on a silver nitrate-impregnatedsilica (20% AgNO₃), with petroleum ether:EtOAc (2:1) as eluent to give0.73 g (60%) of the linoleic acid ethyl ester (>96% purity by GC; GC-MS:MW 309 (GC-MS for a control non-deuterated linoleic acid ethyl ester: MW308). ¹H NMR (400 MHz, CDCl₃) δ 0.89 (t, J=7.0 Hz, 3H, CH₃), 1.25 (t,J=7.0 Hz, 3H, CH₃CH₂O), 1.30 (m, 14H, CH₂), 1.61 (m, 2H, CH₂), 2.04 (m,2H), 2.28 (t, J=7.5 Hz, 2H, CH₂COOEt), 2.74 (m, 1H, CHD), 4.12 (q, J=7.0Hz, 2H, OCH₂CH₃), 5.34 (m, 4H, CH═CH). ¹³C NMR (100 MHz, CDCl₃) δ 14.1,14.2, 22.6, 25.0, 25.3 (t, J=19.5 Hz), 27.17, 27.19, 29.08, 29.09,29.14, 29.3, 29.6, 31.5, 34.4, 60.1, 127.8, 128.0, 130.0, 130.2, 173.9.

To obtain the free [11-²H]-linoleic acid (616), to the solution of thelinoleic acid ethyl ester (0.704 g, 2.3 mmol) in ethanol (10 ml) asolution of KOH (0.4 g, 7.1 mmol) in water (0.8 ml) was added. Themixture was stirred at 50° C. for 10 min and then diluted with water (20ml), treated with 10% solution of sulphuric acid (5 ml) and extractedwith Et₂O (4×20 ml). The combined organic fractions were washed withsaturated NaCl, dried over Na₂SO₄, and the solvent was removed atreduced pressure. The residue was flushed through a small volume ofsilica gel (2 ml; eluent:petroleum ether:EtOAc (2:1)) and the solventremoved in vacuo to yield 0.629 g (98%) of the indicated acid 616. ¹HNMR (400 MHz, CDCl₃) δ 0.88 (t, J=7.0 Hz, 3H, CH₃), 1.30 (m, 14H, CH₂),1.60 (m, 2H, CH₂), 2.03 (m, 4H, CH₂), 2.33 (t, J=7.5 Hz, 2H, CH₂COOEt),2.74 (m, 1H, CHD), 5.32 (m, 4H, CH═CH), 11.6 (br s, 1H, COOH). ¹³C NMR(100 MHz, CDCl₃) δ 14.1, 22.6, 24.6, 25.3 (t, J=19.0 Hz), 27.16, 27.18,29.00, 29.05, 29.12, 29.3, 29.6, 31.5, 34.0, 127.8, 128.0, 130.0, 130.2,180.1.

Example 7 Synthesis of [11-¹³C]-Linoleic Acid

[1-¹³C]-oct-2-yn-1-ol (717).

The title compound has been synthesized according to the earlierdescribed protocols (Hill, Sh.; Hirano, K.; Shmanai, V. V.; Marbois, B.N.; Vidovic, D.; Bekish, A. V.; Kay, B.; Tse, V.; Fine, J.; Clarke, C.F.; Shchepinov, M. S. Free Radic. Biol. Med., 2011, 50 (1), 130-138)using ¹³C-paraform, and used without further purification. ¹H NMR(CDCl₃, δ): 4.22 (

, J=148 Hz, 2H), 2.18 (td, J₁=7.0, J₂=1 Hz, 2H), 1.91 (br s, 1H), 1.47(quint, J=7.0 Hz, 2H), 1.31 (m, 4H), 0.87 (t, J=7.0 Hz, 3H).

[1-¹³C]-1-bromooct-2-yne (718)

was synthesized as described in (Hill, Sh.; Hirano, K.; Shmanai, V. V.;Marbois, B. N.; Vidovic, D.; Bekish, A. V.; Kay, B.; Tse, V.; Fine, J.;Clarke, C. F.; Shchepinov, M. S. Free Radic. Biol. Med., 2011, 50 (1),130-138). Yield: 82% starting from ¹³C-paraform (per two steps). ¹H NMR(CDCl₃, δ): 3.93 (dt, J₁=158 Hz, J₂=2 Hz, 2.23 (m, 2H), 1.50 (m, 2H),1.33 (m, 4H), 0.89 (t, J=7 Hz, 3H).

[11-¹³C]-ethyl octadeca-9,12-diynoate (719).

was synthesized as previously described (See Meyer, M. P.; Klinman, J.P. Tetrahedron Lett. 2008, 49, 3600; Hill, Sh.; Hirano, K.; Shmanai, V.V.; Marbois, B. N.; Vidovic, D.; Bekish, A. V.; Kay, B.; Tse, V.; Fine,J.; Clarke, C. F.; Shchepinov, M. S. Free Radic. Biol. Med., 2011, 50(1), 130-138). Yield: 93%. ¹H NMR (CDCl₃, δ): 4.10 (q, J=7 Hz, 2H), 3.1(dm, J=134 Hz, 2H), 2.27 (t, J=7.5 Hz, 2H), 2.13 (m, 4H), 1.60 (m, 2H),1.47 (m, 4H), 1.3 (m, 10H), 1.24 (t, J=7 Hz, 3H), 0.88 (t, J=7.0 Hz,3H).

[11-¹³C]-linoleic acid ethyl ester (720)

was synthesized as previously described (See Meyer, M. P.; Klinman, J.P. Tetrahedron Lett. 2008, 49, 3600; Hill, Sh.; Hirano, K.; Shmanai, V.V.; Marbois, B. N.; Vidovic, D.; Bekish, A. V.; Kay, B.; Tse, V.; Fine,J.; Clarke, C. F.; Shchepinov, M. S. Free Radic. Biol. Med., 2011, 50(1), 130-138). Yield: 56%. ¹H NMR (CDCl₃, δ): 5.34 (m, 4H), 4.12 (q, J=7Hz, 2H), 2.77 (dm, J=126 Hz, 2H), 2.28 (t, J=7.5 Hz, 2H), 2.04 (m, 4H),1.61 (m, 2H), 1.30 (m, 14H), 1.25 (t, J=7 Hz, 3H), 0.88 (t, J=7.0 Hz,3H).

[11-¹³C]-linoleic acid (721)

was synthesized as previously described (See Meyer, M. P.; Klinman, J.P. Tetrahedron Lett. 2008, 49, 3600; Hill, Sh.; Hirano, K.; Shmanai, V.V.; Marbois, B. N.; Vidovic, D.; Bekish, A. V.; Kay, B.; Tse, V.; Fine,J.; Clarke, C. F.; Shchepinov, M. S. Free Radic. Biol. Med., 2011, 50(1), 130-138); yield 98%. ¹H NMR (CDCl₃, δ): 10.5 (br s, 1H), 5.34 (m,4H), 2.77 (dm, J=126 Hz), 2.33 (t, J=7.5 Hz, 2H), 2.03 (m, 4H), 1.60 (m,2H), 1.30 (m, 14H), 0.88 (t, J=7.0 Hz, 3H).

Example 8 General Preparation of Esters A-D

General Procedure for Compound A.

Thionyl chloride (2 equivalents) is slowly added to a solution of PUFA(1 equivalent) in CHCl₃. The reaction mixture is heated to reflux for 1hr, then it is allowed to cool to room temperature and the solvent isevaporated under reduced pressure to afford the carboxylic acid chloridederivative of the PUFA. The carboxylic acid chloride derivative is thendissolved in anhydrous pyridine and the alcohol (1 equivalent) dissolvedin pyridine is slowly added (Note that the order of addition is reversedwhen the alcohol is a polyalcohol). Upon complete addition, the reactionmixture is allowed to stir at room temperature for 24 hr. The solvent isthen removed under reduced pressure and the crude product is purified bycolumn chromatography to afford Compound A.

11,11-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (30);14,14-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (23);11,11,14,14-Tetradeutero-cis,cis,cis-octadeca-8,12,15-trienoic acid(17); and 11,11-Dideutero-cis,cis-octadeca-9,12-dienoic acid (7) areeach subjected to the above described procedure with the followingalcohols: ethanol, glycerol, propylene glycol; glucose;2-(2-ethoxyethoxy)ethanol; and estradiol to afford productscorresponding to the general formula of Compound A.

General Procedure for Compound B.

Thionyl chloride (2 equivalents) is slowly added to a solution of PUFA(1 equivalent) in CHCl₃. The reaction mixture is heated to reflux for 1hr, then it is allowed to cool to room temperature and the solvent isevaporated under reduced pressure to afford the carboxylic acid chloridederivative of the PUFA. The carboxylic acid chloride derivative is thendissolved in anhydrous pyridine and the alcohol (Compound A, 1equivalent) dissolved in pyridine is slowly added. Upon completeaddition, the reaction mixture is allowed to stir at room temperaturefor 24 hr. The solvent is then removed under reduced pressure and thecrude product is purified by column chromatography to afford Compound B.

The Compound A products that form from the condensation of11,11-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (30);14,14-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (23);11,11,14,14-Tetradeutero-cis,cis,cis-octadeca-8,12,15-trienoic acid(17); and 11,11-Dideutero-cis,cis-octadeca-9,12-dienoic acid (7) withglycerol, propylene glycol; glucose; and estradiol are treated accordingto the above-described general procedure with11,11-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (30);14,14-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (23);11,11,14,14-Tetradeutero-cis,cis,cis-octadeca-8,12,15-trienoic acid(17); and 11,11-Dideutero-cis,cis-octadeca-9,12-dienoic acid (7) as thePUFAs to afford products corresponding to the general formula ofCompound B.

General Procedure for Compound C.

Thionyl chloride (2 equivalents) is slowly added to a solution of PUFA(1 equivalent) in CHCl₃. The reaction mixture is heated to reflux for 1hr, then it is allowed to cool to room temperature and the solvent isevaporated under reduced pressure to afford the carboxylic acid chloridederivative of the PUFA. The carboxylic acid chloride derivative is thendissolved in anhydrous pyridine and the alcohol (Compound B, 1equivalent) dissolved in pyridine is slowly added. Upon completeaddition, the reaction mixture is allowed to stir at room temperaturefor 24 hr. The solvent is then removed under reduced pressure and thecrude product is purified by column chromatography to afford Compound C.

The Compound B products that form from the condensation of Compound Aproducts with glycerol and glucose are treated according to theabove-described general procedure with 11,11-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (30);14,14-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (23);11,11,14,14-Tetradeutero-cis,cis,cis-octadeca-8,12,15-trienoic acid(17); and 11,11-Dideutero-cis,cis-octadeca-9,12-dienoic acid (7) as thePUFAs to afford products corresponding to the general formula ofCompound C.

General Procedure for Compound D.

Thionyl chloride (2 equivalents) is slowly added to a solution of PUFA(1 equivalent) in CHCl₃. The reaction mixture is heated to reflux for 1hr, then it is allowed to cool to room temperature and the solvent isevaporated under reduced pressure to afford the carboxylic acid chloridederivative of the PUFA. The carboxylic acid chloride derivative (4equivalents) is then dissolved in anhydrous pyridine and the alcohol (1equivalent) dissolved in pyridine is slowly added. Upon completeaddition, the reaction mixture is allowed to stir at room temperaturefor 24 hr. The solvent is then removed under reduced pressure and thecrude product is purified by column chromatography to afford Compound D.

The Compound C products that form from the condensation of Compound Bproducts with glucose are treated according to the above-describedgeneral procedure with11,11-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (30);14,14-Dideutero-cis,cis,cis-octadeca-8,12,15-trienoic acid (23);11,11,14,14-Tetradeutero-cis,cis,cis-octadeca-8,12,15-trienoic acid(17); and 11,11-Dideutero-cis,cis-octadeca-9,12-dienoic acid (7) as thePUFAs to afford products corresponding to the general formula ofCompound D.

Example 9 ¹H- and ¹³C-NMR Analysis of Deuterated PUFAs Described inExamples 1-4 (FIG. 2)

Characteristic areas of ¹H and ¹³C spectra, all values in ppm. (Panel A)Deuteration of Lin acid at pos. 11 is confirmed by the disappearance ofpeaks in ¹H and ¹³C NMR spectra. Disappearance of the peak at δ_(H)2.764 is expected due to absence of H atoms (¹H NMR). Disappearance ofthe peak at δ_(C) 25.5 in is due to combination of Nuclear OverhauserEffect, and splitting of this particular carbon atom into a quintet bytwo D atoms in the deuterated form of Lin acid. (Panel B) The ¹H NMRspectrum shows that the H atoms at C11 and C14 positions ofsite-specifically deuterated ΔLnn coincide (δ_(H) 2.801) thusdeuteration at either site (11,11-H₂, 14,14-D₂ or 11,11-D₂, 14,14-H₂)leads to a 50% decrease in integration of this peak, while deuterationof both sites (11,11,14,14-D₄) leads to the complete disappearance ofthe peak at δ_(H) 2.801. However, ¹³C NMR experiments can clearlydistinguish between the three deuterated forms, as the observed peaksfor C11 and C14 positions are separated by a small but detectabledifference. Thus, deuteration at either C11 or C14 positions leads todisappearance of the peak at δ_(C) 25.68 or δ_(C) 25.60, respectively,while deuteration at both sites leads to disappearance of the twocorresponding peaks.

Example 10 Isotope Reinforcement can Shut Down PUFA Peroxidation

Q-less yeast (coq mutants) provide an ideal system to assess in vivoautoxidation of fatty acids. Coenzyme Q (ubiquinone or Q) serves as asmall lipophilic antioxidant as well as an electron shuttle in therespiratory chain of the mitochondrial inner membrane. Ten S. cerevisiaegenes (COQ1-COQ10) are required for coenzyme Q biosynthesis andfunction, and the deletion of any results in respiratory deficiency(Tran U C, Clarke C F. Mitochondrion 2007; 7S, S62). It was shown thatthe coq yeast mutants are exquisitely sensitive to autoxidation productsof PUFAs (Do T Q et al, PNAS USA 1996; 93:7534-7539; Poon W W, Do T Q,Marbois B N, Clarke C F. Mol. Aspects. Med. 1997; 18, s121). Although S.cerevisiae do not produce PUFAs (Paltauf F, Daum G. Meth. Enzymol. 1992;209:514-522), they are able to utilize PUFAs when provided exogenously,allowing their content to be manipulated (Paltauf F, Daum G. Meth.Enzymol. 1992; 209:514-522). Less than 1% of Q-less (coq2, coq3, andcoq5) yeast mutants is viable following a four hour treatment withlinolenic acid (Do T Q et al, PNAS USA 1996; 93:7534-7539; Poon W W, DoT Q, Marbois B N, Clarke C F. Mol. Aspects. Med. 1997; 18, s121). Incontrast, 70% of wild-type (the parental genetic background is strainW303-1B) cells subjected to this treatment remain viable. The Q-lessyeast are also hypersensitive to other PUFAs that readily autoxidize(such as arachidonic acid), but behave the same as the wild-typeparental strain to treatment with the monounsaturated oleic acid (Do T Qet al, PNAS USA 1996; 93:7534-7539). The hypersensitivity of the Q-lessyeast mutants is not a secondary effect of the inability to respire,because cor1 or atp2 mutant yeast (lacking either the bcl complex or theATP synthase, respectively) show wild-type resistance to PUFA treatment(Do T Q et al, PNAS USA 1996; 93:7534-7539; Poon W W, Do T Q, Marbois BN, Clarke C F. Mol. Aspects. Med. 1997; 18, s121).

A plate dilution assay can be used to assess PUFA sensitivity. Thisassay can be performed by spotting serial five-fold dilutions ofaliquots onto YPD plate media (FIG. 3). The sensitivity of the differentstrains can be observed by visual inspection of the density of cells ineach spot.

Treatment with linolenic acid causes a dramatic loss of viability of thecoq null mutants. In stark contrast, coq mutants treated with theD4-linolenic acid were not killed, and retained viabilities similar toyeast treated with oleic acid. Quantitative colony counting revealedthat the viability of cells treated with oleic and D4-linolenic wassimilar (FIG. 4), while the viability of the coq mutants was reducedmore than 100-fold following treatment with the standard linolenic acidfor 4 h. These results indicate that isotope-reinforced linolenic acidis much more resistant to autoxidation than is the standard linolenicacid, as evidenced by the resistance of the hypersensitive coq mutantsto cell killing.

Example 11 GC-MS Can Detect Fatty Acids and PUFAs in Yeast Cells

Yeast do not synthesize PUFAs, however they do incorporate exogenouslysupplied linoleic and linolenic acids (Avery S V, et al. AppliedEnviron. Microbiol. 1996; 62,3960; Howlett N G, et al. Applied Environ.Microbiol. 1997; 63,2971). Therefore, it seems likely that yeast wouldalso incorporate exogenously supplied D4-linolenic acid. However, it ispossible that the differential sensitivity to linolenic and D4-linolenicmight be attributed to differences in integration into the cell ratherthan autoxidation. To test whether this is the case, the extent ofuptake of this fatty acid was monitored. First the conditions ofseparation of fatty acid methyl esters (FAME) of C18:1, C18:3, D4-18:3and C17:0 (to be used as an internal standard) were determined. TheGC-MS chromatogram shown in FIG. 5 establishes both separation andsensitivity of detection of these fatty acid methyl ester standards.

Wild-type yeast were harvested during log phase growth and incubated inthe presence of exogenously added fatty acid (for 0 or 4 h) in thepresence of phosphate buffer plus 0.20% dextrose, as described for thefatty acid sensitivity assay. Cells were harvested, washed twice with 10ml sterile water, and the yeast cell pellets were then processed byalkaline methanolysis as described above. The fatty acids are detectedas methylesters (FAMEs) following GC-MS with C17:0 added as an internalstandard (FIG. 6). The amounts of 18:3 and D4 detected after 4 hincubation were extrapolated from the calibration curve. These resultsindicate yeast avidly incorporate both linolenic and D4-linolenic acidduring the 4 h incubation period. Based on these results, it is obviousthat the enhanced resistance of the coq mutant yeast to treatment withD4-C18:3 is not due to lack of uptake.

D2-linolenic, 11,11-D2-linolenic acid and 14, 14-D2-linolenic acid, werealso used on this yeast model and rendered comparable protection.

Example 12 Kinetic Isotope Effect in Non-Enzymatic Oxidation of D2-LA ina Chain Reaction Format

The kinetics of oxygen consumption during the oxidation of LA and D2-LAwas studied with a glass capillary microvolumeter (FIG. 7). The rate ofoxidation, R_(OX), was measured as a slope of [O₂]traces. The rate ofinitiation, R_(IN), was determined by the inhibitor method with HPMC(“6-hydroxy-2,2,5,7,8-pentamethylbenzochroman”) as a referenceinhibitor. R_(IN) was calculated from the induction period of inhibitedoxidation, t_(IND): R_(IN)=2·[HPMC]/t_(IND). The rate of oxidation of0.71 M LA (FIG. 7) was found to be 6.1×10⁻⁶ M/s. When the process wasinhibited by 0.23 mM chain-breaking antioxidant HPMC, the duration ofthe induction period, t_(IND), was about 48 min, with the R_(IN) valueof around 0.16×10⁻⁶ M/s. The length of the kinetic chain calculated fromthese data was: ν=R_(OX)/R_(IN)=38±3. Based on this data, the calculatedoxidizability of LA was 0.0215±0.008 M^(−0.5) s^(−0.5) (n=5) [CosgraveJ. P, et. al. Lipids, 1987, 22, 299-304]. For D2-LA, the reduction ofR_(OX) to 0.18×10⁻⁶ M/s was observed (FIG. 7). In contrast to LA,addition of HPMC did not result in the decrease in R_(OX) and theappearance of any detectable induction period (data not shown). Thelatter precludes a direct determination of R_(IN). For a R_(IN) valuefor D2-LA oxidation being comparable to that of LA it follows that D2-LAoxidation was not a chain process (ν=0.18×10⁻⁶/0.16×10⁻⁶≈1.1). Anestimated kinetic isotope effect (“KIE”), from comparison of R_(OX) forLA and D2-LA, was around 6.1×10⁻⁶/0.18×10⁻⁶≈35. A similar KIE wasdetermined during the oxidation of LA and 11,11-d₂-LA in Triton X-100aqueous micelles (data not shown). For comparative purposes, thetheoretical KIE is 6.9 at 25° C. See Carpenter, “Determination ofOrganic Reaction Mechanisms” (John Wiley & Sons, 1984), p. 89.

Example 13 Small Amounts of D2-LA Protect LA Against Peroxidation

To simulate the likely in vivo conditions, the kinetics of the oxidationof the mixtures of D2-LA and LA were studied (FIG. 8). In theexperiments, the concentration of LA plus 11,11-d2-LA was 0.775 M; theconcentration of AMVN was 0.0217 M; and the reactions were carried outat 37° C. The results afforded an R_(IN) of 1.10±0.08×10⁻⁷ M/sec.Additionally, the rate of oxidation of the mixtures was found to benon-additive and much lower than the additive value of R_(OX) for theindividual compounds. Surprisingly, D2-LA essentially ‘protects’ thenon-deuterated LA against autoxidation. A qualititatively similar effectwas also observed during the oxidation of the mixture of 11,11-D2-LAwith non-deuterated methyl linoleate (data not shown). These resultssuggest that even a partial replacement of non-deuterated LA by D2-LAmay substantially slow down PUFA peroxidation.

Example 14 Small Amounts of D2-LA Protect LA Against Peroxidation InVivo

The results described in Example 13 were reproduced in vivo using Q-lesscoq3 yeast strains and different ratios of LA to D2-LA (FIG. 9).Wild-type, yeast Q-less coq3, or respiratory deficient cor1 null mutantswere incubated in the presence of 200 μM of LA and D2-LA at differentratios of PUFAs, as indicated in FIG. 9. Serial dilutions (1:5) startingat 0.2 OD/ml were spotted on YPD solid plate medium. Additionally, azero-time untreated control was utilized and the results are shown onthe top left of FIG. 9. Growth was at 30° C. The results indicate thatapproximately 10-15% of D2-LA was a sufficiently minimal amount tocancel the toxicity of LA. A similar incubation with the mono-deuteratedPUFA, 11,11-D,H-LA, afforded no detectable loss in cell viability after3 hours of treatment (data not shown). These results suggest that bothD2-LA and 11,11-D,H-LA were resistant to lipid peroxidation.

Wild-type yeast cells were treated as described above except the yeastwere treated with 200 μM of the designated fatty acid for 2 hours,washed with sterile water, and were either not treated (triangles) ortreated with 50 μM CuSO₄ (squares) at room temperature. After 60 min ofcopper treatment cells were treated with 8 μM C11-Bodipy 581/591 for 30min at room temperature. Four 100 μl aliquots were plated in a 96-wellplate and the fluorescence was measured. Wild-type yeast cells treatedwith copper in the absence or presence of PUFA have significantly higherlevels of lipid peroxidation as compared to yeast not treated withcopper. However, copper-stressed wild-type yeast cells treated with11,11-D₂-LA have lower levels of lipid peroxidation similar to yeast nottreated with PUFA. Mono-deuterated 11,11-D,H-LA offered similarprotection.

Example 15 Small Amounts of D4-ALA Protect ALA Against Peroxidation InVivo

The experimental protocol described for Example 14 was also reproducedin vivo using Q-less coq3 yeast strains (FIG. 10) and different ratiosof ALA to D4-ALA. Wild-type, yeast Q-less coq3, or respiratory deficientcor1 null mutants were incubated in the presence of 200 μM of ALA andD4-Lnn (Linolenic acid) at different ratios of PUFAs, as indicated inFIG. 10. Serial dilutions (1:5) starting at 0.2 OD/ml were spotted onYPD solid plate medium. Growth was at 30° C. The results indicate thatapproximately 15-20% of D2-Lnn was a sufficiently minimal amount tocancel the toxicity of ALA. Moreover, results indicate that the contentof PUFA taken up by yeast cells roughly reflects the ratios added andsuggests that yeast cells do not discriminate among the PUFAs provided.

Example 16 D-PUFA Mitigates Oxidative Stress and Increases Survival inRetinal Cells Implicated in AMD and Diabetic Retinopathy Pathology

Several cell types, including microvascular endothelium (MVEC), retinalpigment epithelium (RPE) and retinal neurons (retinal ganglion cells)were tested for survival in cell culture. Cells were kept in the mediumcontaining either hydrogenated (control) or deuterated D2-linoleic (ω-6;LA) and D4-linolenic (ω-3; ALA) acids (20 μM; ratio of ω-6 to ω-3:1:1 or2:1) for 72 hrs. The incorporation of PUFAs into cells was monitored byGC (FIG. 11). PUFAs were shown to be readily taken up by cells accordingto the Table 1, showing incorporation of PUFAs into MVECs.

TABLE 1 Area unlabelled Area labelled ratio control linoleate 783929764556042 0.058 linolenate 1488866 149411 0.100 PUFA linoleate 960268305525295 0.058 linolenate 2347729 113468 0.048 Deuterated PUFA linoleate34957060 2599969 0.074 linolenate 747128 134824 0.180

The cells were then treated with paraquat (PQ; 500 μM), a commonoxidative stress-generating compound. For survival measurement, cellswere counted using haemocytometer and trypan blue exclusion method. FIG.12 shows the survival of H- and D-PUFA treated MVEC cells after acuteintoxication by paraquat. For all cell types tested, D-PUFA hadprotective effect compared to controls, similar to that shown in FIG. 8for MVEC cells.

Example 17 Toxicology Studies of Mice Supplemented with D-PUFA Reveal NoAnomalies in Major Blood Biomarkers

With a more protracted dosing paradigm (i.e. 3 weeks of dietaryreplacement), chemical analysis of blood serum of H-PUFA- andD-PUFA-supplemented mice (performed at UC Davis) revealed no differencein major biomarkers of renal function, liver function, blood lipids, etcfor H-PUFA/D-PUFA saline treated mice. In this example, D-PUFA is a 2:1mixture of D2-linoleic acid: D4-linolenic acid.

Tested parameters included measurements of triglycerides; total protein;total bilirubin; phosphorus; free fatty acids; HDL; glucose; creatine;cholesterol; calcium; blood urea nitrogen; alkaline phosphatase;albumin; aspartate aminotransferase; and others in Table 2.

TABLE 2 Alanine Aspartate Alkaline Blood Urea Mouse SampleAminotransferase Aminotransferase Albumin Phosphatase Nitrogen CalciumCholesterol ID # volume U/L U/L g/dl U/L mg/dl mg/dl mg/dl  4 100 273.03008.7 3.09 81.7 19.1 7.96 148.3  5 110 5726.7 8478.9 3.42 31.1 25.47.40 185.1  7 100 156.0 1470.6 2.82 35.1 18.9 7.64 151.2 10 60 518.44653.0 3.02 QNS 20.1 6.78 184.0 11 70 144.0 1635.3 3.63 72.7 20.3 8.75170.8 13 14 3518.1 15669.0 QNS <0.1 31.5 QNS 166.5 14 75 216.9 2107.83.03 42.4 24.4 7.46 173.6 25 75 589.5 4707.0 3.20 18.8 18.0 5.97 193.427 100 727.2 6015.6 2.63 <0.1 36.2 5.71 166.7 28 100 468.9 4018.5 2.9349.3 21.2 6.90 164.4 29 29 1898.1 12510.0 QNS QNS 24.9 QNS 208.8 30 1002963.7 5371.2 3.38 50.3 18.2 6.29 174.7 Mean 76 1508 5289 3.17 52.6 22.87.67 168.5 D-PUFA SD 33 2225 5189 0.30 23.0 4.6 0.66 14.5 D-PUFA Mean 811329 6524 3.04 39.5 23.7 6.22 181.6 H-PUFA SD 31 1078 3428 0.33 17.9 80.51 19.0 D-PUFA High Density Non-esterified Total Total MouseCreatinine Glucose Lipoprotein Fatty Acid Phosphorus Bilirubin ProteinTriglyceride ID # mg/dl mg/dl mg/dl mEq/L mg/dl mg/dl g/dl mg/dl  40.189 160.2 104.49 1.08 13.07 0.185 5.32 38.9  5 0.356 355.6 134.37 1.0718.59 0.275 6.56 57.9  7 0.154 174.6 107.39 1.11 10.14 0.192 5.26 82.710 0.151 136.5 138.15 1.06 QNS 0.272 6.07 46.1 11 0.179 107.9 139.861.18 9.33 0.162 5.72 33.5 13 1.126 176.4 135.09 0.99 QNS QNS QNS 31.5 140.170 93.3 47.78 1.06 10.41 0.235 6.07 43.8 25 0.126 164.5 147.96 1.0118.39 0.269 6.74 41.0 27 1.453 88.3 98.46 0.87 24.57 0.301 6.26 26.9 280.232 224.9 50.54 1.02 14.16 0.231 5.87 49.6 29 0.111 QNS 77.58 0.20 QNSQNS QNS 27.9 30 0.225 227.4 131.04 1.17 21.42 0.349 6.28 46.7 Mean 0.332172.1 115.30 1.08 12.31 0.220 5.83 47.8 D-PUFA SD 0.357 87.0 33.21 0.063.78 0.048 0.50 17.7 D-PUFA Mean 0.429 176.3 101.12 0.85 19.64 0.2886.29 38 H-PUFA SD 0.575 65.5 39.40 0.38 4.44 0.050 0.36 11 D-PUFA

Example 18 Histopathologic Studies

Microscopic changes were coded by the most specific topographic andmorphologic diagnosis, and the Systematized Nomenclature of Medicine(SNOMED) and the National Toxicology Program's Toxicology DataManagement System (TDMS) terminology manuals were used as guidelines.Data were recorded in Labcat® Histopathology module 4.30. A four-stepgrading system (minimal, mild, moderate, and marked) was used to definegradable changes.

C57BL6 male mice were dosed orally in the diet with PUFAs on Study Days1 through 14, and were necropsied on Study Day 15. Group 1 consisted of4 mice and received hydrogenated PUFAs. Group 2 consisted of 5 mice andreceived deuterated PUFAs (D2-LA and D4-ALA) On Study Day 8, all micereceived intraperitoneal (IP) saline. Complete sets ofprotocol-specified tissues [liver (3 to 7 sections), lungs with bronchi(2 to 5 lobes), spleen, heart, and kidneys] from all submitted mice wereexamined histopathologically. No difference was observed between theH-PUFA and D-PUFA groups.

Example 19 Evaluation of Tissue-Specific Deuteration

WT mice were housed at 12 animals (males separate from females) per cageand fed for 90 days ad libitum (typically, 5-6 g/day) on the AIN 93diet, as pellets, with 6% total fat. Approximately 10% of that total fatwas made up of 1:1 mixture of D2-LA/D4-ALA (group 1), D2-LA/ALA (group2), or LA/ALA (control group). The animals were sacrificed, organsharvested and stored at low temperature prior to analysis without theuse of preservation agents. Lipid fractions were separated, pre-treatedand analyzed by LC-MS according to standard protocols using LA, D2-LA,ALA and D4-ALA as controls.

Dosage studies of 1:1 D2-LA/D4-ALA indicated that tissues became highlyenriched in deuterium, with about 40% of the total fat being deuterated(FIG. 13). Moreover, these studies indicated that fat distributionremained relatively unchanged by the tested dosage (FIG. 14). Afterdosage studies of 1:1 D2-LA/ALA, it was determined that about 27% of thetotal fat was deuterated (FIG. 15).

Specific organs, such as the liver and brain, were also evaluated (FIGS.16-21). While the liver had a different fat profile than previoustissues studied (FIG. 16), 90 day dosage studies with D2-LA/D4-ALAdemonstrated that tissues became highly enriched in deuterium, withabout 40% of the total fat being deuterated (FIG. 17). Moreover, theliver study indicated that fat distribution remained relativelyunchanged by the tested dosage (FIG. 16-17). Additionally, 90 day dosagestudies with D2-LA only illustrated a similar fat profile as previousstudies, along with about 32% total fat being deuterated (FIG. 18).Consequently, fat profiles and deuteration profiles in the liver weremaintained regardless of the administered deuterated component. Like theliver, the brain also had a different fat profile than previous tissuesstudied (FIGS. 19-21). 90 day dosage studies with D2-LA/D4-ALAdemonstrated that tissues became highly enriched in deuterium, withabout 30% of the total fat being deuterated (FIG. 19). Moreover, thebrain study indicated that fat distribution remained relativelyunchanged by the tested dosage (FIGS. 19-21). Additionally, 90 daydosage studies with D2-LA/ALA illustrated a similar fat profile asprevious studies, along with about 23% total fat being deuterated (FIG.20). Consequently, fat profiles and deuteration profiles in the brainwere maintained regardless of the administered deuterated component.

Example 20 Testing PUFA Incorporation into Eye Tissues and Cells

Isotope ratio Mass-spectrometry can be used to confirm incorporation ofD-PUFA into the phospholipid membranes of eye tissues. When deliveringD2-LA and D4-ALA through dietary supplementation, incorporation intoanimal tissues can be monitored using an isotope ratio mass-spectrometrytechnique that will allow for measurement of the total increase indeuterium composition in lipid membranes, thus reporting onincorporation of D2-LA, D4-ALA, and any other PUFA derived from thesecompounds. Using this method, a substantial uptake of D-PUFA into mouseeye tissue can be detected. For example, mice are supplemented withD-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg/kg of D2-LA, D4-ALA, and 1:1combinations of both D2-LA and D4-ALA) or H-PUFA (0.01, 0.1, 1.0, 10.0,and 100 mg/kg of LA, ALA, and 1:1 combinations of both LA and ALA) asthe only PUFA source for 6 days, exposed acutely to a known oxidant orsaline vehicle and continued on the same diet for an additional 6 days.Eyes are removed and dissected, and homogenate samples fromsaline-treated mice and test compound-treated mice are analyzed fordeuterium content as described for Example 7 above. D2-LA and D4-ALA areexpected to be found in the tissues and cells analyzed.

Example 21 Testing for Efficacy Against Age-Related Macular Degeneration

Retinal pigment epithelium (RPE) cells from human donor eyes arecultured according to known methods and exposed to D-PUFA (0.01, 0.1,1.0, 10.0, 100, and 1000 μM D2-LA, D4-ALA, and 1:1 combinations of bothD2-LA and D4-ALA) or H-PUFA (0.01, 0.1, 1.0, 10.0, 100, and 1000 μM LA,ALA, and 1:1 combinations of both LA and ALA). At several time points(24, 28, and 72 hours), digital images are taken and analyzed withrespect to lipofuscin and pigmentation according to known methods. D2-LAand D4-ALA are expected to prevent formation of lipofuscin.

The treatment-dependent ability of RPE cells to phagocytose, which is acrucial function, can also be evaluated. Retinal pigment epithelium(RPE) cells from human donor eyes are cultured according to knownmethods and exposed to D-PUFA (0.01, 0.1, 1.0, 10.0, 100, and 1000 μMD2-LA, D4-ALA, and 1:1 combinations of both D2-LA and D4-ALA) or H-PUFA(0.01, 0.1, 1.0, 10.0, 100, and 1000 μM LA, ALA, and 1:1 combinations ofboth LA and ALA). After approximately three weeks, fluorescent latexbeads are added to the cell cultures for approximately four hours. Thecells could are washed to remove non-phagocytosed beads and fixed.Fluorescent images of the cells are taken and analyzed for lipofuscincontent and the number of phagocytosed beads. D2-LA and D4-ALA areexpected to prevent formation of lipofuscin yet allow for the cells toretain their ability to phagocytose.

The effects of lipofuscin in vivo, i.e. in the living eye ofexperimental rats after an intravitreal injection of the compounds canalso be investigated. D-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg/kg D2-LA,D4-ALA, and 1:1 combinations of both D2-LA and D4-ALA) or H-PUFA (0.01,0.1, 1.0, 10.0, and 100 mg/kg LA, ALA, and 1:1 combinations of both LAand ALA) are injected intravitreally in half-year old Wistar rats. Therats are then be evaluated using electroretinography for functionaltesting and by counting lipofuscin particles. D2-LA and D4-ALA areexpected to decrease lipofuscin content.

An animal model of dry AMD (See Justilien V, Pang, J P, et al, SOD2Knockdown Mouse Model of Early AMD, Invest Opthalmol V is Sci, 2007, 48(10), 4407-4420) is known which uses mice that have had a ribozyme thattargets the protective enzyme manganese superoxide dismutase (MnSOD)injected beneath the retina. The ribozyme (AAV-Rz 432) method wouldallow for site specific somatic knockdown of SOD 2 expression in normaladult tissue so that the lesion is only in the injected eye and the restof the body is unaffected. Thus the animal's other eye can act as acontrol. This protocol has been shown to induce in the eyes of the miceafter a single subretinal injection of the ribozyme many of theabnormalities seen in the eyes of patients with AMD, with the mostsevere changes seen 120 days post injection. The surrogate markers forprotection from reactive oxygen and nitrogen species damage can beexamined by electroretinography (ERG) and morphometry.

For example, full field ERG analysis may be used to assess the loss ofrod and cone function and measuring a-wave and b-wave amplitudes in darkadapted and light adapted mice. Digital fundus imaging may be used tomonitor atrophic changes in the retinal pigment epithelium (RPE).Additionally, light microscopy may be used to qualitatively assessdamage to the photoreceptors and RPE, to look for evidence of choroidalneovascularization and to measure changes in the thickness of Bruch'smembrane. Retinal degeneration is determinable morphometrically bymeasuring the thickness of the outer nuclear layer and rod outersegments (See Goto Y, Peachey N S, Ripps H, Naash M I. Functionalabnormalities in transgenic mice expressing a mutant rhodopsin gene,Invest Opthalmol V is Sci 1995, 36, 62-71; Faktorovich E G, Steinberg RH, Yasumura D, Matthes M T, LaVail M M. Photoreceptor degeneration ininherited dystrophy delayed by the basic fibroblast growth factor,Nature 1990, 347, 83-86).

Accordingly, D-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg/kg D2-LA, D4-ALA,and 1:1 combinations of both D2-LA and D4-ALA) or H-PUFA (0.01, 0.1,1.0, 10.0, and 100 mg/kg LA, ALA, and 1:1 combinations of both LA andALA) are administered to the mouse model of dry AMD described inJustilien V, Pang, J P, et al, SOD2 Knockdown Mouse Model of Early AMD,Invest Opthalmol V is Sci, 2007, 48 (10), 4407-4420). The surrogatemarkers for protection from reactive oxygen and nitrogen species damageare examined by electroretinography (ERG) and morphometry. D2-LA andD4-ALA are expected to protect against damage caused by reactive oxygenand nitrogen species.

As described above, new blood vessel growth and leakage are thought tofacilitate wet AMD disease progression. As a representative model,artificial corneal burns can be induced in rat eyes to determine theeffects of vehicle solution, non-stabilized test compounds, or testcompounds at various concentrations on corneal neovascularization. Morespecifically, topical administration of vehicle solution, non-stabilizedtest compounds, or test compounds at various concentrations could beadministered twice a day to rats in which corneal burns had beenartificially induced by application of silver nitrate (70%) and/orpotassium nitrate (30%).

Accordingly, D-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg/kg D2-LA, D4-ALA,and 1:1 combinations of both D2-LA and D4-ALA) or H-PUFA (0.01, 0.1,1.0, 10.0, and 100 mg/kg LA, ALA, and 1:1 combinations of both LA andALA) are daily administered to rats having artificial corneal burns.Over a three week period, the rats are examined for new blood vesselgrowth and leakage. At the end of the trial, the rats are euthanized,their eyes are dissected, and blood vessel growth and leakage arefurther examined. D2-LA and D4-ALA are expected to decrease the amountof new vessel growth and leakage.

Example 22 Testing for Efficacy Against Diabetic Retinopathy

Diabetes mellitus occurs in spontaneously diabetic Torii (SDT) rats atabout 20 weeks of age. Accordingly, blood glucose levels can beconfirmed at about 25 weeks of age, and rats showing an increase inblood glucose level (at least 300 mg/dl) can be used as a model fordetermining a compound's effectiveness at preventing and/or mitigatingthe diabetic retinopathy. For example, male SDT rats that developdiabetes mellitus are divided into two groups, that is, 1) a controlgroup and 2) a test compound treatment group. D-PUFA (0.01, 0.1, 1.0,10.0, and 100 mg/kg D2-LA, D4-ALA, and 1:1 combinations of both D2-LAand D4-ALA) or H-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg/kg LA, ALA, and1:1 combinations of both LA and ALA) are mixed with the rats' usual feedcontrolled for LA and ALA and fed daily. At 36 weeks of age and at 52 to58 weeks of age, the rats are anesthetized and their eyeballs areexcised and used in histopathological examination.

The histopathological examination is carried out in the followingmanner. The rat is anesthetized with ether and by intraperitonealadministration of Nembutal, and then the eyeballs are excised. Theexcised eyeballs are placed in a mixed solution (1:1:2) consisting of 4%glutaraldehyde, 10% neutral formalin and a phosphate buffer (pH 7.2, 0.3mol/l). After 60 minutes, the eyeball is cut into halves under astereoscopic microscope and then stored overnight at 4° C. The nextmorning, the eyeballs are embedded in a usual manner into paraffin toprepare a transverse section containing a bundle of optic nerves. Thesection is then stained with hematoxylin-eosin and analyzed according toknown methods. D2-LA and D4-ALA are expected to prevent the developmentof retinopathy.

Fluorescein fundus angiography is also carried out by opening the rats'chests under the same anesthesia, injecting fluorescein dextran (Sigma,50 mg/l ml PBS) into the heart, and after 5 minutes, excising theeyeballs. The retina is then separated from the excised eyeball, spreadon a slide glass to prepare a flat mounted specimen of the retina, andobserved with a stereoscopic fluorescent microscope and photographed toevaluate retinopathy.

If retinal fold (retinal detachment) accompanied by thickening of theretina around the optic nerve head is recognized in the pathologicalexamination, or when fluorescence dye leakage accompanied by retinalvascular tortuosity and/or caliber variation around the optic nerve headis recognized in the fluorescein fundus photography, it is assumed thatsevere retinopathy occurred in the SDT rat.

Example 23 Testing for Efficacy Against Stargardt Disease

ABCR^(−/−) mice is an animal model of human recessive Stargardt'sDisease. As in humans with recessive Stargardt's disease, ABCR^(−/−)mice accumulate large lipofuscin deposits in the RPE cells of their eyesand eventually experience delayed dark adaptation. Lipofuscinaccumulation and vision loss observed in this mouse model is alsothought to be relevant to age-related macular degeneration (AMD) andother macular dystrophies.

ABCR^(−/−) mice are raised on a diet of D-PUFA (0.01, 0.1, 1.0, 10.0,and 100 mg/kg D2-LA, D4-ALA, and 1:1 combinations of both D2-LA andD4-ALA) or H-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg/kg LA, ALA, and 1:1combinations of both LA and ALA). After 1, 3, 5, and 7 weeks, mice aresacrificed, their eyes are dissected, and the retinas and eyecups arepooled and homogenated with ethanol. The homogenate is centrifuged, thesupernatant is drawn off, and the supernatant is analyzed forA2E-lipofuscin by HPLC. D2-LA and D4-ALA are expected to impedelipofuscin formation.

Example 24 Testing for Efficacy Against Retinitis Pigmentosa

Because α-phosphodiesterase-related mutations in RP-patients play animportant part in degeneration, the rd-1 mouse is a relevant and usablemodel for determining the efficacy of the compounds disclosed herein. Inuntreated rd-1 mice the mutation brings about apoptosis of rod receptorcells which begins on about postnatal day 10 (PN 10), and eventuallyleads to night blindness on about PN18. At PN18 only cone photoreceptorcells remain, which will also eventually die. For comparative purposes,normal wild-type mice which are genetically the same as the rd-1 mousecould be used.

Test animals could be treated in accordance with the European Communityguideline (86/609/EEC). Mice of the CH3 line and homozygous retinaldegeneration 1 (rd1/rd1) are used. On the third day following birth(PN3) test animals are treated with D-PUFA (0.01, 0.1, 1.0, 10.0, and100 mg/kg D2-LA, D4-ALA, and 1:1 combinations of both D2-LA and D4-ALA)or H-PUFA (0.01, 0.1, 1.0, 10.0, and 100 mg/kg LA, ALA, and 1:1combinations of both LA and ALA) for two weeks. Animals are sacrificedone day after completion of the treatment period, whereafter their eyesare removed and analysed for the number of photoreceptors in on eachside of the optic nerve using the so-called “Terminal dUTP nick-endlabelling-assay (TUNEL).” Commercially available cell death detectionkits are used to quantify the assay. D2-LA and D4-ALA are expected toprotect against photoreceptor cell death.

Experiments are also performed on an organ culture of retina explantsfrom rd-1 and control mice under serum-free conditions, as described inCaffe A R, Ahuja P, Holmqvist B, Azadi S, Forsell J, Holmqvist I,Soderpalm A K, van Veen T (2001) “Mouse retina explants after long-termculture in serum-free medium.”, J Chem Neuroanat 22: 263-273, which isincorporated herein by reference. D2-LA and D4-ALA are expected toprotect against cell death.

CONCLUSION

While the invention has been described with reference to the specificembodiments thereof, it should be understood by those skilled in the artthat various changes may be made and equivalents may be substitutedwithout departing from the true spirit and scope of the invention. Thisincludes embodiments which do not provide all of the benefits andfeatures set forth herein. In addition, many modifications may be madeto adapt a particular situation, material, composition of matter,process, process step or steps, to the objective, spirit and scope ofthe present invention. All such modifications are intended to be withinthe scope of the claims appended hereto. Accordingly, the scope of theinvention is defined only by reference to the appended claims.

What is claimed is:
 1. A method of treating or preventing theprogression of an oxidative retinal disease, comprising: administeringan effective amount of a polyunsaturated substance to a Wet or DryAge-related Macular Degeneration (AMD), Retinitis Pigmentosa (RP),Diabetic Retinopathy (DR), Cataracts, or Stargardt Disease (SD) patientin need of treatment, wherein the polyunsaturated substance ischemically modified such that one or more bonds is stabilized againstoxidation; wherein the polyunsaturated substance or a polyunsaturatedmetabolite thereof comprising said one or more stabilized bonds isincorporated into the patient's body following administration.
 2. Themethod of claim 1, wherein the polyunsaturated substance is a fattyacid, a fatty acid mimetic, or a fatty acid pro-drug.
 3. The method ofclaim 2, wherein the fatty acid, fatty acid mimetic, or fatty acidpro-drug is stabilized at one or more bis-allylic positions.
 4. Themethod of claim 3, wherein the stabilization comprises at least one ¹³Catom or at least one deuterium atom at a bis-allylic position, whereinthe at least one ¹³C atom or the at least one deuterium atom is presentat a level significantly above the naturally-occurring abundance levelof said isotope.
 5. The method of claim 4, wherein the stabilized fattyacid, fatty acid mimetic, or fatty acid pro-drug comprise between about10% and 50% of the total amount of fatty acids, fatty acid mimetics, orfatty acid pro-drugs administered to the patient.
 6. The method of claim4, wherein the isotopically stabilized fatty acid, fatty acid mimetic,or fatty acid pro-drug comprise between about 10% and 30% of the totalamount of fatty acids, fatty acid mimetics, or fatty acid pro-drugsadministered to the patient.
 7. The method of claim 4, wherein theisotopically stabilized fatty acid, fatty acid mimetic, or fatty acidpro-drug comprise about 20% or more of the total amount of fatty acids,fatty acid mimetics, or fatty acid pro-drugs administered to thepatient.
 8. The method of claim 4, wherein a cell or tissue of thepatient maintains a sufficient concentration of the fatty acid, fattyacid mimetic, or fatty acid pro-drug to prevent autooxidation of thenaturally occurring polyunsaturated fatty acid, mimetic, or esterpro-drug.
 9. The method of claim 4, wherein the polyunsaturatedsubstance is an omega-3 fatty acid, fatty acid mimetic, or fatty acidpro-drug, or an omega-6 fatty acid, fatty acid mimetic, or fatty acidpro-drug.
 10. The method of claim 9, wherein the polyunsaturatedsubstance is selected from the group consisting of 11,11-D2-linolenicacid, 14,14-D2-linolenic acid, 11,11,14,14-D4-linolenic acid,11,11-D2-linoleic acid, 14,14-D2-linoleic acid, 11,11,14,14-D4-linoleicacid, 11-D-linolenic acid, 14-D-linolenic acid, 11,14-D2-linolenic acid,11-D-linoleic acid, 14-D-linoleic acid, and 11,14-D2-linoleic acid. 11.The method of claim 9, wherein the polyunsaturated substance is furtherstabilized at a pro-bis-allylic position.
 12. The method of claim 4,wherein the polyunsaturated substance is a fatty acid pro-drug ester.13. The method of claim 12, wherein the ester is a triglyceride,diglyceride, or monoglyceride.
 14. The method of claim 2 furthercomprising co-administering an antioxidant.
 15. The method of claim 14,wherein the antioxidant is Coenzyme Q, idebenone, mitoquinone,mitoquinol, vitamin C, or vitamin E.